Phosphoramidate derivatives of 5-fluoro-2′-deoxyuridine for use in the treatment of cancer

ABSTRACT

Phosphoramidate derivates of 5-fluoro-2′-deoxyuridine are disclosed for use in the treatment of cancer, especially in the treatment of cancer where the patient shows resistance, for example, in a patient with cells with a lowered level of nucleoside transporter proteins and/or with nucleoside kinase-deficient cells and/or with mycoplasma-infected cells and/or with cells with a raised level of thymidylate synthase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/489,884, filedApr. 18, 2017, which is a continuation of U.S. Ser. No. 14/943,555,filed Nov. 17, 2015, now issued U.S. Pat. No. 9,655,915, which is acontinuation of U.S. Ser. No. 14/560,097, filed Dec. 4, 2014, now issuedU.S. Pat. No. 9,221,866 which is a continuation of U.S. Ser. No.14/000,682, filed Nov. 14, 2013, issued U.S. Pat. No. 8,993,053 which isa 35 USC 371 national stage application of International App. No.PCT/GB2012/050457, filed Feb. 29, 2012, which claims priority to GB1103582.1, filed Mar. 1, 2011, and GB 1105660.3, filed Apr. 1, 2011.

The present invention relates to chemical compounds useful in thetreatment of cancer.

In 1957, the antitumour activity of 5-Fluorouracil (5FU) was discovered.More than fifty years since it was first synthesised, 5FU remains widelyused in the treatment of solid tumours including breast,gastrointestinal system, head, neck and ovary and in particular ofcolorectal cancer, as approved by FDA in 1962. The fluoropyrimidine5-fluorouracil (5FU) and 5-fluoro-2′-deoxyuridine (5-FdUrd) are used incombination with folic acid as standard treatment for a variety ofcarcinomas, as stomach, colon and breast. Moreover, a combination of 5FUwith leucovorin (LV) is considered as standard chemotherapy for coloncancer. The drug 5FU is usually administered by intravenous bolus or bycontinuous infusion.

The antitumour activity of 5FU is comparable to that of its analogue5-FdUrd, which partly acts as prodrug of 5FU. 5-FdUrd was approved byFDA in 1970, and has been used extensively for the clinical treatment ofcarcinoma of the ovary, breast and gastrointestinal tract. Moreover dueto extensive hepatic extraction 5-FdUrd is a useful drug for hepaticarterial chemotherapy of liver metastases thereby it is more efficientlymetabolized by the liver than 5FU.

A problem exists, however, in that activity of both the agents 5FU and5-FdUrd can be impaired by the development of resistance in tumourcells. Treatment of cancer with 5FU has also been found to causeneurotoxic and cardiotoxic side effects. Toxicity also derives from thelack of selectivity of 5FU towards tumours.

It is an object of the present invention to provide compounds derivedfrom 5-fluoro-2′-deoxyuridine that show an enhanced activity and/orreduced toxicity in their treatment of cancer, compared to that shown by5-fluoracil or 5-fluoro-2′-deoxyuridune per se.

It is a further object of the present invention to provide compoundsderived from 5-fluoro-2′-deoxyuridine that show a low level ofresistance in tumour cells, in particular a resistance in tumour cellsthat is less than that shown by 5FU or by 5-FdUrd.

According to the present invention there is provided a compound offormula (I):

whereinAr is a fused bicyclic aryl moiety or a monocyclic aryl moiety, eitherof which aryl moieties is carbocyclic or heterocyclic and is optionallysubstituted;R₃ is alkyl, which is optionally substituted;R₄ is H or alkoyl; andR₁ and R₂ are independently selected from the group consisting of H andalkyl or R₁ and R₂ together form an alkylene chain so as to provide,together with the C atom to which they are attached, a cyclic system, orone of R₁ and R₂ comprises an alkylene chain attached to N, the H atomattached to N is absent and one of R₁ and R₂ comprises H or alkyl, anyof which alkyl moieties or alkylene chains may be substituted;or a pharmaceutically acceptable derivative or metabolite of formula I,wherein the compound is not a compound having, in combination, Ar asunsubstituted phenyl, R₃ as CH₃, R₄ as H and one of R₁ and R₂ as H andone of R₁ and R₂ as CH₃.

It has been found that the compounds of the present invention showactivity that renders them useful in the prophylaxis or treatment ofcancer in Homo sapiens. In particular, the present compounds exhibitbeneficial properties which indicate their ability to treat cancer inpatients whilst showing reduced resistance in tumour cells. Notably,compounds of the present invention can show a cytoactivity comparable toor better than that of 5-fluoracil, but with a resistance that iscomparable to or less than that of each of 5-fluoracil and5-fluoro-2′-deoxyuridine.

By “resistance” in the present application is meant a low or diminishedresponse to therapy. Resistance can be innate or acquired. An innateresistance is a reduced responsiveness relative to other specimens orpatients. An acquired resistance is a reduced effectiveness over thecourse of time in a given patient, whether or not acquired inconjunction with therapy comprising the administration to the patient ofa drug regime to treat cancer, for example, a drug regime comprising 5FUand/or 5-FdUrd. Each of innate resistance and acquired resistance cancorrespond to the downregulation or low activity of transporterproteins, including nucleoside transporter proteins, or necessaryanabolic enzymes or the upregulation of catabolic enzymes.

Although the applicant does not wish to be bound by any theory, it ispostulated, as discussed further below, that causes of resistance intumour cells to the activity of 5FU and/or 5-FdUrd could be: a) deletionof activating kinase as thymidine kinase (TK), a key enzyme required forthe initial phosphorylation step from 5-FdUrd to 5-FdUMP; b)overproduction of thymidylate synthase (TS); and/or c) deficienttransport into target cells.

Surprisingly it has now been found that compounds of the presentinvention can show significant cytostatic activity in cells with alowered level of nucleoside transporter proteins and/or with nucleosidekinase-deficient cells and/or in mycoplasma-infected cells.

The beneficial property of compounds of the present invention to retainmarked cytostatic activity in nucleoside kinase-deficient cells mayconfer in vivo a clinical advantage in cellular environments lacking innucleoside kinases or having decreased levels of nucleoside kinases andthus unable to efficiently activate 5-FdUrd.

Mycoplasma-infected cells greatly reduce the activity of nucleosidessuch as 5-FdUrd due, it is believed, to the overproduction ofthymidylate synthase (TS). The presently proposed use of the presentcompounds in mycoplasma-infected cells thus, it is postulated, derivesfrom the beneficial property of the present compounds to actadditionally as a TS inhibitor and so permit the present compounds toretain their cytostatic activity in mycoplasma-infected cells. Theprodrugs comprising the compounds of the present invention, due to theirlipophylic nature may be taken up by the target cells in an at leastpartially nucleoside transport carrier-independent way, and thus, maycircumvent potential resistance mechanisms due to lowered levels ofnucleoside or nucleobases transport carriers in the target cellmembrane.

Additionally, the prodrugs comprising the compounds of the presentinvention are surprisingly insensitive to the action of the catabolicenzyme Thymidine Phosphorylase (TP) that is often upregulated in tumors,and thus, the prodrugs would be more independent of the presence of thiscatabolic enzyme than 5-FdUrd.

It has been observed that mycoplasma infection of cells can greatlyreduce the activity of nucleosides, including 5-FdUrd. Administration ofa TP inhibitor restores the cytostatic activity of 5-FdUrd inmycoplasma-infected cell cultures, providing evidence of thedeteriorating role of TP in the eventual cytostatic activity of 5-FdUrd.This may be a limitation in patients that are mycoplasma infected.Unlike 5-FdUrd, the 5-FdUrd prodrugs of the present invention can retainhigh activity in these mycoplasma-infected cells.

The present compounds thus have the potential to overcome many of thelimitations of 5-FU and 5-FdUrd.

5-Fluorouracil (5FU) is one of the first examples of an anticancer drug.The design of 5-FU was based on the available biochemical information: afluorine atom and a hydrogen atom have a similar size, however acarbon-fluorine bond is much stronger than a carbon-hydrogen bond.Thymidylate synthase acts by replacing the 5-hydrogen of deoxyuridinemonophosphate with a methyl group obtained from methylenetetrahydrofolate to make thymidylate. 5FU exerts its cytotoxic effectthrough three different pathways. The nucleobase 5FU and thedeoxyribonucleoside 5-FdUrd enter cells through facilitated nucleosidetransport systems. One of the mechanisms of action of these agents isinhibition of the enzyme thymidylate synthase (TS). The nucleobase 5FUis converted to the deoxynucleoside 5-fluoro-2′-deoxyuridine (5-FdUrd)by thymidine phosphorylase.

Subsequent phosphorylation of the deoxynucleoside 5-FdURd by thymidinekinase results in formation of the cytotoxic nucleotide5-fluoro-2′-deoxyuridine-5′-monophosphate (5-FdUMP). In the presence ofthe reduced folate, 5,10-methylene-tetrahydrofolate (mTHF), thenucleotide (5-FdUMP) inhibits thymidylate synthase (TS) due to theinability of the enzyme to remove the 5-fluorine atom. Thus, the firstand the foremost important mechanism of action of 5FU and FDUR isinhibition of the enzyme thymidylate synthase (TS). Thymidylate synthase(TS) has two substrates for (dUMP and mTHF), both of which bind in thecatalytic site to enable the synthesis of dTMP. 5-FdUMP forms a covalentternary complex with thymidylate synthase (TS), inhibiting this enzymeactivity and leading to depletion of deoxythymidine triphosphate,necessary for DNA synthesis. Alternatively, (5-FdUMP) is synthesizedafter conversion of 5FU to 5-FUMP by OPRT, to fluorouridine diphosphate(FUDP), fluorodeoxyuridine diphosphate (5-FdUDP) by ribonucleotidereductase (RR) and eventually to 5′-FdUMP. It has been observed thatafter drug exposure to 5FU or 5-FdUrd, the cells develop resistance tothese chemotherapeutic agents. The overexpression of thymidylatesynthase (TS) reduces the therapeutic effect of TS inhibitory drugleading to resistance. It was observed that some individuals are moreresistant to TS targeted therapy than others. Secondly, thedeoxynucleoside 5-fluoro-2′-deoxyuridine (5-FdUrd) can be converted toits triphosphate 5-FdUTP form which in turn can be incorporated into DNAcausing cell damage. Thirdly, 5FU may also inhibit RNA synthesis by itsconversion to FUMP by OPRT and subsequently, in two steps, tofluorouridine triphosphate (FUTP) that is incorporated into RNA. This isbelieved to be another potential action of 5FU.

The molecule 5FU thus does not result in an optimal TS inhibitory drugbecause it is inefficiently converted to 5-FdUMP due to the severalmetabolic steps required for metabolic activation of 5FU. Furtherresistance can occur if the cell produces excess quantities of dUMP tocompete with the drug for the active site.

5-FdUrd is a relatively good substrate for thymidine kinase, whichconverts it directly to 5-FdUMP. In vitro studies, in several cancercell lines have demonstrated that 5-FdURd is about 5000 fold more potentas inhibitor of cell growth than 5FU. Furthermore, the prodrug 5-FdURdshows no significant conversion to ribonucleotide metabolites atcytotoxic concentrations. In vivo studies showed that a significantamount of 5-FdUrd is degraded to its relative base 5FU by thymidinephosphorylase, enzyme for which 5-FdUrd shows a good affinity. Thisrapid phosphorolytic cleavage of 5-FdUrd to 5FU in vitro and in vivorepresents a major obstacle in delivering intact 5-FdUrd to cells forenhanced cytotoxic action. In addition, the degradation of 5-FdUrd inrat intestinal homogenates and in humans, after oral administration,suggests that 5-FdUrd would scarcely be absorbed as intact 5-FdUrd.

According to a further aspect of the present invention, the compound ofthe present invention is provided for use in a method of prophylaxis ortreatment of cancer in Homo sapiens. Suitably, the cancer is selectedfrom the group comprising leukemia, pancreatic, prostate, lung, breastand cervical cancer.

In particular, the compound of the present invention is for use in amethod of prophylaxis or treatment of cancer in a patient who hasdeveloped or has the potential to develop resistance in tumour cellswith respect to the activity of 5-fluoracil or 5-fluoro-2′-deoxyuridinein the prophylaxis or the treatment of cancer. For example, the compoundof the present invention can be for use in a method of prophylaxis ortreatment of cancer in a patient with cells with a lowered level ofnucleoside transporter proteins and/or with nucleoside kinase-deficientcells and/or with mycoplasma-infected cells, particularly where thecancer is leukemia. The compound of the present invention can instead ofor as well as be for use in a method of prophylaxis or treatment ofcancer in a patient who has cells with a raised level of thymidylatesynthase (TS).

According to a further aspect of the present invention, there isprovided a method of propylaxis or treatment of cancer comprisingadministering to a Homo sapiens patient in need of such treatment aneffective dose of a compound of the present invention. Suitably thecancer is selected from the group comprising leukemia, pancreatic,prostate, lung, breast and cervical cancer.

In particular, the present invention comprises a method for treating apatient who has developed or has the potential to develop resistance intumour cells with respect to the activity of 5-fluoracil or5-fluoro-2′-deoxyuridine in a method of prophylaxis or treatment ofcancer. For example, the method of the present invention can comprisetreating a patient with cells with a lowered level nucleosidetransporter proteins and/or with nucleoside kinase-deficient cellsand/or with mycoplasma-infected cells, particularly where the cancer isleukemia. The method of the present invention for treating a patient caninstead of or as well as be for treating a patient that has cells with araised level of thymidylate synthase (TS).

“Tumour” or “tumour cell” as used in the present application, unlessotherwise indicated, refers to both solid tumours and cancers such asleukemia.

Compounds of the present invention can be used for treating a patientwith cancer either alone de novo or in conjunction with other cancertherapy. For example, compounds of the present invention can be used ina cancer treatment regime in conjunction with other anti-cancer drugs,such as 5-FU and/or 5-FdUrd either, with or without leucovorin (LV),and/or other anti-cancer drugs. Alternatively, compounds of the presentinvention can be used where a patient has failed to respond to otheranti-cancer drugs, such as for example 5FU and/or 5-FdUrd either with orwithout leucovorin (LV), or where the patient has shown resistance toother anti-cancer drugs, such as for example 5-FU and/or 5-FdUrd eitherwith or without leucovorin (LV).

Compounds of the present invention where Ar is 1-naphthyl, whethersubstituted or unsubstituted, are particularly suitable for use in theabove uses and methods of the present invention, particularly in apatient who has developed, or who has the potential to develop,resistance in tumour cells, such as, for example, a patient with cellswith a lowered level of nucleoside transporter cells and/or withkinase-deficient cells and/or with mycoplasma-infected cells and/or apatient who has cells with a raised level of thymidylate synthase (TS).

According to a further aspect of the present invention, there isprovided a pharmaceutical composition comprising a compound of thepresent invention in combination with a pharmaceutically acceptablecarrier, diluent or excipient.

According to another aspect of the present invention, there is provideda method of preparing a pharmaceutical composition comprising the stepof combining a compound of the present invention with a pharmaceuticallyacceptable excipient, carrier or diluent.

According to another aspect of the present invention, there is provideda process for the preparation of a compound of the present inventioncomprising reacting a compound of formula (II)

with a compound of formula (III)

wherein Ar, R₃, R₄, R₁ and R₂ have the meanings described above and inclaim 1.

The group Ar comprises a substituted or unsubstituted aryl group,wherein the term “aryl group” and the possible substitution of saidgroup is as defined herein. Suitably, Ar is a 5 to 14 membered aromaticring moiety. Preferably, Ar is carbocyclic. Alternatively, the one ortwo rings may include 1, 2, 3 or 4 heteroatoms, preferably 1, selected,independently, from O, S and N. Preferably, Ar is a fused carbobicyclicaryl moiety. More preferably, Ar is naphthyl, even more preferably1-naphthyl i.e. naphthyl linked to P via O bonded at the 1-naphthylposition. Suitably, Ar can alternatively be phenyl.

One, two, three or four substituents, which may be the same ordifferent, may be present on Ar and are selected from the groupcomprising halogen, which may —F, —Cl, —Br or —I; —NO₂; —NH₂; optionallysubstituted —C₁₋₃alkyl; optionally substituted —C₁₋₃alkoxy, preferablymethoxy (—OCH₃); optionally substituted —SC₁₋₃alkyl; —CN; optionallysubstituted —COC₁₋₃alkyl; and optionally substituted —CO₂C₁₋₃alkyl;where said optionally substituted groups may be substituted with one ormore up to six, preferably three, members independently selected fromthe group comprising halogen, which may be F, Cl, Br and I, and NO₂.Particularly preferred substituents on Ar are electron withdrawinggroups such as halogen (preferably chlorine or fluorine), trihalomethyl(preferably trifluoromethyl), cyano and nitro groups.

The substituents may be at any position on the Ar aryl moiety. Where Aris 1-naphthyl, a single substituent at any of positions 2, 3, 4, 5, 6, 7or 8 is preferred. Where Ar is phenyl, a single substituent at the 2(ortho) or 4 (para) position is preferred, more preferred at the 4position. For example, where Ar is a substituted phenyl, Ar can be3,5-dichloro-phenyl, p-trifluoromethyl-phenyl, p-cyano-phenyl, orp-nitro-phenyl.

Suitably, R₃ is a C₁₋₁₆ primary, secondary or tertiary alkyl group andcan include carbocyclic moieties; a C₅₋₇ cyclic alkyl group; or aC₁₋₆alkylC₅₋₁₁aryl group. More suitably, R₃ is a C₁₋₁₀ alkyl group orC₁₋₃ alkylC₅₋₇ aryl group such as benzyl (—CH₂—C₆H₅). A cyclic alkylgroup can be carbocyclic or can contain, in total, one, two or threering heteroatoms selected independently from O, N and S. Preferably R₃is unsubstituted. Where substituted, substituents are set out below.

Suitably R₄ is H or alkoyl i.e. alkyl-C(═O)—, where alkyl is C₁ to C₁₀.

When R₁ and/or R₂ is alkyl, they are each independently suitablyselected from C₁ to C₁₆, more suitably from C₁ to C₆. When R₁ and R₂together comprise an alkylene chain, the chain is suitably C₁ to C₆ andmay contain unsaturation and, in total, one, two or three heteroatoms inthe chain independently selected from O, N and S. When one of R₁ and R₂is attached to N, the total ring size including N and the C atom towhich R₁ and R₂ are attached suitably comprises 4 to 7 members, moresuitably 5 members. Any alkyl or alkylene chain comprising R₁ and/or R₂can be substituted with one or more substituents set out herein.

When R₁ and R₂ are different, the C atom to which they are attached ischiral. Preferably, the stereochemistry at an asymmetric centre —CR₁R₂corresponds to an L-amino acid. The stereochemistry at an asymmetriccentre —CR₁R₂ can, however, correspond to a D-amino acid. Alternatively,mixtures of compounds can be employed having asymmetric centrescorresponding to L and D amino acids.

Suitably, R₁ and R₂ can correspond to the moieties attached to the alphaC atom in a naturally occurring alpha amino acid. By “naturallyoccurring alpha amino acid” is meant Alanine, Arginine, Asparagine,Aspartic Acid, Cysteine, Cystine, Glycine, Glutamic Acid, Glutamine,Histidine, Hydroxylysine, Hydroxyproline, Isoleucine, Leucine, Lysine,Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan,Tyrosine and Valine. One of R₁ and R₂ can thus be H and one of R₁ and R₂can thus be H or alkyl selected from the following moieties or R₁ and R₂together can form an alkylene chain selected from the followingmoieties:

CH₃— as present in Alanine H₂NC(═NH)NH[CH₂]₃— as present in ArgenineNH₂C(O)CH₂— as present in Aspargine HO₂CH₂— as present in Asparctic AcidHSCH₂— as present in Cysteine HO₂CH(NH₂)CH₂SSCH₂— as present in CystineH— as present in Glycine HO₂CH₂CH₂— as present in Glutamic AcidH₂N(O)CCH₂CH₂— as present in Glutamine C₃N₂HCH₂— as present in HistidineH₂NCH₂CH(OH)CH₂CH₂— as present in Hydroxylysine —CH₂CH(OH)CH₂— aspresent in Hydroxyproline CH₃CH₂CH(CH₃)— as present in Isoleucine(CH₃)₂CHCH₂— as present in Leucine H₂NCH₂(CH₂)₃— as present in LysineCH₃SCH₂CH₂— as present in Methionine PhCH₂— as present in Phenylalanine—CH₂CH₂CH₂— as present in Proline OHCH₂— as present in Serine CH₃CH(OH)—as present in Threonine C₈NH₆CH₂— as present in Tryptophan HOC₆H₄CH₂— aspresent in Tyrosine (CH₃)₂CH— as present in Valine.

By “a pharmaceutically acceptable derivative” is meant anypharmaceutically acceptable salt, ester, salt of such ester, hydrate,solvate, or crystalline form or metabolite or any other compound whichupon administration to a recipient is capable of providing (directly orindirectly) a compound of formula (I).

Reference in the present specification to an alkyl group means abranched or unbranched, cyclic or acyclic, saturated or unsaturated(e.g. alkenyl or alkynyl) hydrocarbyl radical. Where cyclic, thealkylene group is preferably C₃ to C₁₂, more preferably C₅ to C₁₀, morepreferably C₅ to C₇. Where acyclic, the alkyl group is preferably C₁ toC₁₆, more preferably C₁ to C₆.

Reference in the present specification to an aryl group means anaromatic group containing, suitably, 5 to 14 ring atoms. For example Aris phenyl or naphthyl. The aromatic group may be a heteroaromatic groupcontaining one, two, three or four, preferably one, heteroatomsselected, independently, from the group consisting of O, N and S.Examples of such heteroaromatic groups include pyridyl, pyrrolyl,furanyl and thiophenyl.

The alkyl and aryl groups may be substituted or unsubstituted. Wheresubstituted, there will generally be one to three substituents present,preferably one substituent. Substituents may include halogen atoms, bywhich is meant F, Cl, Br and I atoms, and halomethyl groups such as CF₃and CCl₃; oxygen containing groups such as oxo, hydroxy, carboxy,carboxyC₁₋₁₆alkyl, alkoxy, alkoyl, alkoyloxy, aryloxy, aryloyl andaryloyloxy; nitrogen containing groups such as amino, C₁₋₆alkylamino,diC₁₋₆alkylamino, cyano, azide and nitro; sulphur containing groups suchas thiol, C₁₋₆alkylthiol, sulphonyl and sulphoxide; heterocyclic groupswhich may themselves be substituted; alkyl groups as defined above,which may themselves be substituted; and aryl groups as defined above,which may themselves be substituted, such as phenyl and substitutedphenyl. Substituents on said heterocyclic, alkyl and aryl groups are asdefined immediately above. Substituents in R₁ and/or R₂ include moietiesto provide compounds in which R₁ and R₂ correspond to the moietiesattached to the alpha C atom in a natural occurring alpha amino acid.

Reference in the present specification to alkoxy and aryloxy groupsmeans, respectively, alkyl-O— (for example where alkyl is C₁ to C₁₆,preferably C₁ to C₆) and aryl-O— (for example where aryl is a 5 to 14membered aromatic mono- or bifused ring moiety, optionally containing 1,2, 3 or 4 heteroatoms selected, independently, from O, S and N,preferably aryl is phenyl).

Reference in the present specification to alkoyl and aryloyl groupsmeans, respectively, alkyl-CO— (for example where alkyl is C₁ to C₁₆,preferably C₁ to C₆) and aryl-CO— (for example where aryl is a 5 to 14membered aromatic mono or bifused ring moiety, optionally containing 1,2, 3 or 4 heteroatoms selected, independently, from O, S and N,preferably aryl is phenyl).

Reference in the present specification to alkoyloxy and aryloyloxymeans, respectively, alkyl-CO—O (for example where alkyl is C₁ to C₁₆,preferably C₁ to C₆) and aryl-CO-O (for example where aryl is a 5 to 14membered mono- or bifused aromatic ring system, optionally containing 1,2, 3 or 4 heteroatoms selected, independently, from O, S and N,preferably aryl is phenyl).

Reference in the present specification to heterocyclic groups meansgroups containing one or more, pyrrolyl, imidazolyl, pyraziolyl,thiazolyl, isothiazolyl, oxazolyl, pyrrolidinyl, pyrrolinyl,imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl,pyronly, pyridyl, pyrazinyl, pyridazinyl, piperidyl, piperazinyl,morpholinyl, thionaphthyl, benzofuranyl, isobenzofuryl, indolyl,oxyindolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl,isoindazolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolyl,isoquinolyl, naphthridinyl, cinnolinyl, quinazolinyl, pyridopyridyl,benzoxazinyl, quinoxadinyl, chromenyl, chromanyl, isochromanyl andcarbolinyl.

In one embodiment of the present invention, Ar is suitably naphthyl,especially 1-naphthyl i.e. naphthyl linked to P via O bonded at the1-naphthyl position.

In another embodiment of the present invention, Ar is suitably phenyl.

In one embodiment of the present invention, Ar is substituted. Suitablesubstituents are set out herein.

In one embodiment of the present invention, Ar is unsubstituted1-naphthyl.

In one embodiment of the present invention, Ar is unsubstituted phenyl.

In one embodiment of the present invention, R₄ is selected from thegroup consisting of H and acetyl (CH₃C(═O)—), especially R₄ is H.

In one embodiment of the present invention, R₃ is selected from thegroup consisting of benzyl and members of the group comprising C₁ to C₁₀alkyls, especially R₃ is selected from n-propyl, n-butyl, n-pentyl andn-hexyl, more especially R₃ is n-pentyl.

In one embodiment of the present invention, R₁ and R₂ correspond to themoieties attached to the alpha C atom in a naturally occurring alphaamino acid, as set out herein. A particularly suitable naturallyoccurring alpha amino acid is L-alanine such that suitably one of R₁ andR₂ is H, one of R₁ and R₂ is CH₃ and the C atom to which they areattached has L chirality. In other embodiments, R₁ and R₂ correspond tothe moieties attached to the alpha C atom in a non-naturally occurringalpha amino acid, for example R₁ and R₂ are both suitably CH₃.

The specific features mentioned in the above embodiments arespecifically disclosed to be combined together in any and allcombinations in compounds of the present invention.

Particularly suitable compounds of the present invention are compoundswhere Ar is 1-naphthyl, R₃ is benzyl, one of R₁ and R₂ is H, one of R₁and R₂ is methyl and the C atom to which R₁ and R₂ are attached hasL-chirality and compounds where Ar is 1-naphthyl, R₃ is n-pentyl, one ofR₁ and R₂ is H, one of R₁ and R₂ is methyl and the C atom to which R₁and R₂ are attached has L-chirality. For each compound, R₄ is mostsuitably H.

Conventional treatment of cancer using chemotherapeutics is largelybased on the use of nucleoside analogues. These molecules are designedto mimic natural pyrimidine and purine nucleosides. After uptake by thecell, they are phosphorylated by cellular enzymes such as(deoxy)cytidine kinase (dCK), thymidine kinase (TK) and/ornucleo(s)(t)ide kinases. These antimetabolites can subsequentlyinterfere with the de novo synthesis of DNA/RNA precursors to eventuallyinhibit DNA/RNA synthesis resulting in cytotoxic/static activity (Hatseet al., 1999; Galmarini et al., 2002).

Fluoropyrimidine-based antimetabolites such as fluorouracil (5-FU),capecitabine and 5-fluoro-2′-deoxyuridine (5-FdUrd) are mainly used inthe treatment of colon, breast and ovarian carcinoma (de Bruin et al.,2006; Ishikawa et al., 1998; Walko et al., 2005). Intracellularly, thesedrugs are metabolised to 5-FdUMP, which forms a stable inhibitorycomplex with thymidylate synthase (TS) and the reduced co-substrate5,10-methylenetetrathydrofolate, thereby blocking binding of the normalsubstrate dUMP to the enzyme (Beck et al., 1994; Tanaka et al., 2000;Longley et al, 2003). TS is the enzyme responsible for the conversion ofdUMP to TMP and is therefore indispensible for cell proliferation,making it an interesting target for drug design. Among thefluoropyrimidines mentioned above, 5-FdUrd requires only one metabolicconversion, a phosphorylation catalysed by TK to generate 5-FdUMP(Longley et al., 2003). This obligatory phosphorylation is often therate-limiting step in the metabolism of many anti-cancer drugs(including 5-FdUrd), and is therefore still one of the limiting factorsfor the therapeutic use of nucleoside analogues. Hence, differentstrategies to improve the antitumour efficacy of nucleoside analogueshave been investigated (Galmarini et al., 2002).

The charged nature of nucleoside monophosphates under physiologicalconditions results in poor, if any, penetration across the cell membrane(Mehellou et al., 2009). Therefore, the direct administration ofphosphorylated molecules to circumvent the first phosphorylation stephas little therapeutic advantage. Hence, different strategies forbypassing the rate-limiting phosphorylation using various types ofnucleoside 5′-monophosphate prodrugs for more efficient drug-deliveryhave been explored (Hecker & Erion, 2008). The administration oflipophilic phosphoramidate nucleotide prodrugs (ProTides) has provedsuccessful for several molecules with anti-viral/cancer activity (Harriset al., 2001; Congiatu et al., 2006; McGuigan et al., 2010). By maskingthe charges of the phosphate motif, good passive membrane diffusion ofthe prodrugs can be accomplished after which the prodrug is rapidlyconverted intracellularly into the nucleoside monophosphate by enzymaticcleavage (Mehellou et al., 2009).

Mycoplasmas are the smallest self-replicating organisms on earth and arecharacterized by the lack of a cell wall and a strongly reduced genome(600-1,200 kb). Many of these bacteria have a parasitic lifestyle andreside in the human body causing asymptomatic infections (Razin et al.,1998). It was shown that these prokaryotes tend to preferentiallycolonize tumour tissue: Huang et al. (2001) reported that 39.7-56% ofhuman gastric, colon, oesophageal, lung and breast cancers are infectedwith mycoplasmas compared to 20.9-30% in non-tumourigenic tissue.Pehlivan et al. (2005) found >80% of kidney tissue samples of patientssuffering renal cell carcinoma to be infected with mycoplasmas compared15 to 14% in control tissue samples. Chan et al. (1996) reported a 59%infection rate in ovarian cancer tissues and other studies also report ahigh mycoplasma infection rate in gastric (Sasaki et al., 1995, Yang etal., 2010) and cervical condyloma tissues (Kidder et al., 1998). Due totheir reduced set of genes, mycoplasmas lack the pathway for de novopyrimidine and purine synthesis and therefore express a wide array ofsalvage nucleo(s)(t)ide-metabolizing enzymes, such as thymidinephosphorylase (TP), deoxycytidine deaminase, etc. (Razin, 1978; Charron& Langelier, 1981; Neale et al., 1983; Tham et al., 1993). Already in1985 it was observed that mycoplasma-encoded enzymes (e.g. TP), presentin contaminated cell cultures, lead to decreased dTTP incorporation inlymphocytes (Sinigaglia & Talmadge, 1985). Recently, it has beendemonstrated that these enzymes, in particular the mycoplasma-encodedthymidine phosphorylase, also interfere with the cytostatic activity ofseveral chemotherapeutics, including 5-trifluorothymidine, in vitro(Bronckaers et al., 2008; Jett6 et al., 2008; Liekens et al., 2009).Therefore it has been hypothesized that the elimination of mycoplasmasby antibiotics or suppression of mycoplasma-encoded enzymes in humantumour tissue may optimize treatment of cancer patients using purine andpyrimidine antimetabolites (Liekens et al., 2009).

The present invention is derived from the development and assessment ofTK-independent phosphoramidate prodrugs of 5-FdUrd and providescompounds that can also be insensitive to the TP-dependent inactivationof its free nucleoside analogue. Compounds of the present invention canthus provide mycoplasma-insensitive nucleoside analogue prodrugs whichmay optimize treatment of cancer patients using a pyrimidineantimetabolite. From among the presently synthesized phosphoramidateprodrugs of 5-FdUrd, CPF-373 (identified below and mentioned above as aparticularly suitable compound of the invention with R₄ as H) was chosenfor further in depth studies. This molecule contains a naphthyl andbenzylalaninyl group to mask the charged 5′-phosphate on 5-FdUMP.

Various mechanisms of tumour cell resistance towards fluoropyrimidinessuch as 5FU, 5-FdUrd and trifluorothymidine (TFT) have been described,including a decreased activity of crucial drug-activating enzymes (e.g.TK and orotate phosphoribosyltransferase), an increased activity ofdrug-inactivating enzymes (i.e. thymidine phosphorylase) and/or anupregulation of the target enzymes (e.g. TS) (Agarwal et al., 1999;Murakami et al., 2000; Kosaka et al., 2004). Also, high TP levels foundin several types of cancer tissue were reported to be predictive of apoorer prognosis upon treatment with fluoropyrimidines (Kamoshida etal., 2005; Ciaparrone et al., 2006; Koopman et al., 2009), althoughother studies have not confirmed these findings (Ciccolini et al., 2004;Koopman et al., 2009). The present invention derives from thedevelopment of a prodrug for 5-FdUrd, to circumvent possible resistancemechanisms and susceptibility to degradation by catabolic enzymes,present in the tumour micro-environment.

Compounds embodying the present invention, for example CPF-373, arephosphoramidate prodrugs of 5-FdUrd and are described herein and canfulfil these aims. After uptake into the tumour cells, CPF-373, forexample, generates 5-FdUMP intracellularly upon enzymatic cleavage.Stability studies and enzymatic/serum studies by ³¹P NMR technologyrevealed that the prodrug CPF-373, for example, is fully stable in acidand alkaline conditions, but subject to hydrolysis in the presence ofserum or carboxypeptidase Y, resulting in the formation of thenucleoside 5′-phosphoramidate derivative. Whereas TK is a key enzyme inthe activation of 5-FdUrd, CPF-373, for example, was found to be muchless dependent on TK to exert its cytostatic action in both murine(L1210) and human (CEM) cell cultures. Due to the lipophilic nature ofProTides, these molecules can deliver nucleoside-monophosphates directlyinto the intact tumour cell after conversion to their nucleosidephosphoramidate derivative by enzymes such as carboxyesterases orcarboxypeptidases (i.e. carboxypeptidase Y), eliminating the need for aninitial phosphorylation by specific nucleoside kinases such as TK. Inthis regard, CPF-373, for example, may be an adequate tool for thetreatment of tumour cells with a modified TK activity (be it acquired orinherent). Also, since TK expression is S-phase-dependent, it isexpected that CPF-373, for example, can also efficiently deliver 5-FdUMPin tumour cells that are not in the S-phase of their replication cycle.TS activity studies revealed that, CPF-373, for example, was able toinhibit TS in both wild-type and TK-deficient tumour cell lines,pointing again to an efficient intracellular delivery of the5′-monophosphate of 5-FdUrd, and its virtual independence of cellular TKfor metabolic activation.

Compounds of the present invention, such as CPF-373, are unlikely to beinactivated by catabolic enzymes involved in nucleoside metabolism.Indeed, whereas 5-FdUrd is highly susceptible to enzymatic hydrolysis byTP resulting in the formation of 5-FU and 2-deoxyribose-1-phosphate, itsprodrug, for example CPF-373, is not a substrate for prokaryotic (i.e.E. coli) or mammalian (i.e. human erythrocyte) TP. Also, uridinephosphorylase does not recognize, for example CPF-373, as a substrate,whereas 5-FdUrd is (poorly, but measurably) hydrolyzed by this enzyme.Several studies revealed that many tumour cells have elevated levels ofTP, which also acts as an angiogenic factor (Koopman et al., 2009;Bronckaers et al., 2009). Moreover, there are several reports on thepreferential colonization of tumour tissue by mycoplasmas (Sasaki etal., 1995; Chan et al., 1996; Huang et al., 2001; Pehlivan et al., 2005)which interfere with the cytostatic activity of several conventionalchemotherapeutics in vitro through its encoded TP (Bronckaers et al.,2008; Jett6 et al., 2008; Liekens et al., 2009). The presentobservations that 5-FdUrd, but not, for example, CPF-373, markedly losescytostatic activity when the tumour cells are infected by(TP-expressing) mycoplasmas, is in full agreement with theseobservations. Therefore, the administration of a TP-insensitiveanti-cancer prodrug such as CPF-373, demonstrated to be chemicallystable at extreme pH conditions, may further improve cancerchemotherapy. In conclusion, ProTides, such as CPF-373, provide aninteresting new approach towards the development of more resilientanti-cancer drugs. For instance CPF-373 may have at least severaladvantages over its parent drug 5-FdUrd: it exerts its cytostaticactivity independent of TK and it is resistant to metabolic breakdown byTP, an enzyme that is often upregulated in tumours or may be externallyexpressed by mycoplasma infection of the tumour tissue.

The compound having formula I or the pharmaceutical compositionaccording to the present invention can be administered to a Homo sapienspatient by any suitable means.

The medicaments employed in the present invention can be administered byoral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration.

For oral administration, the compounds of the invention will generallybe provided in the form of tablets or capsules, as a powder or granules,or as an aqueous solution or suspension.

Tablets for oral use may include the active ingredient mixed withpharmaceutically acceptable excipients such as inert diluents,disintegrating agents, binding agents, lubricating agents, sweeteningagents, flavouring agents, colouring agents and preservatives. Suitableinert diluents include sodium and calcium carbonate, sodium and calciumphosphate, and lactose, while cornstarch and alginic acid are suitabledisintegrating agents. Binding agents may include starch and gelatin,while the lubricating agent, if present, will generally be magnesiumstearate, stearic acid or talc. If desired, the tablets may be coatedwith a material such as glyceryl monostearate or glyceryl distearate, todelay absorption in the gastrointestinal tract.

Capsules for oral use include hard gelatin capsules in which the activeingredient is mixed with a solid diluent, and soft gelatin capsuleswherein the active ingredient is mixed with water or an oil such aspeanut oil, liquid paraffin or olive oil.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the compounds of the invention will generally be provided in sterileaqueous solutions or suspensions, buffered to an appropriate pH andisotonicity. Suitable aqueous vehicles include Ringer's solution andisotonic sodium chloride. Aqueous suspensions according to the inventionmay include suspending agents such as cellulose derivatives, sodiumalginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agentsuch as lecithin. Suitable preservatives for aqueous suspensions includeethyl and n-propyl p-hydroxybenzoate.

The compounds of the invention may also be presented as liposomeformulations.

In general a suitable dose will be in the range of 0.1 to 300 mg perkilogram body weight of the recipient per day. A preferred lower dose is0.5 mg per kilogram body weight of recipient per day, a more preferredlower dose is 6 mg per kilogram body weight of recipient per day, aneven more preferred lower dose is 10 mg per kilogram body weight perrecipient per day. A suitable dose is preferably in the range of 6 to150 mg per kilogram body weight per day, and most preferably in therange of 15 to 100 mg per kilogram body weight per day. The desired doseis preferably presented as two, three, four, five or six or moresub-doses administered at appropriate intervals throughout the day.These sub-doses may be administered in unit dosage forms, for example,containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably50 to 700 mg of active ingredient per unit dosage form.

Examples of the present invention will now be described, by way ofexample only, with reference to the accompanying drawings comprisingFIGS. 1 to 11, wherein:

FIG. 1 shows structural formula of 5-FdUrd and its phosphoramidateprodrug CPF-373;

FIGS. 2(A), 2(B), and 2(C) show the effect of thymidine phosphorylaseand uridine phosphorylase on dThd, Urd, 5-FdUrd and CPF-373, where dataare the mean of at least 2 independent experiments (±S.D.);

FIGS. 3(A), 3(B), 3(C), 3(D), 3(E), and 3(F) show the inhibition of TSby 5-FdUrd and CPF-373 as measured by tritium release from [5-³H]dUrd(panels A and B) and [5-³H]dCyd (panels C and D) in L1210/0 cellcultures and by tritium release from [5-³H]dCyd (panels E and F) inL1210/TK⁻ cell cultures, where data are the mean of 2 independentexperiments (±S.E.M.);

FIG. 4 shows a proposed putative mechanism of activation of 5-FdUrdProTides;

FIG. 5 shows carboxypeptidase-mediated cleavage of prodrug CPF-373monitored by 31P NMR;

FIG. 6 shows ³¹P NMR spectrum of compound CPF-373 in serum;

FIG. 7 shows ³¹P NMR spectrum of compound CPF-373 in buffer pH=1;

FIG. 8 shows ³¹P NMR spectrum of compound CPF-373 in buffer pH=8;

FIGS. 9(A) and 9(B) show spectra of nucleoside and relative base by ¹⁹FNMR: a) 5-FdUrd submitted to the phosphorylase assay (A); b) 5-FdUrd andthe base 5FU under condition of the assay in absence of the enzyme (TP)(B);

FIGS. 10(A) and 10(B) show spectra of nucleoside and base in potassiumphosphate buffer (205 nM) by ¹⁹F NMR: a) 5-FdUrd submitted to thephosphorylase assay in absence of enzyme (A); b) Result after theaddition of enzyme (TP) (B); and

FIG. 11 shows spectra of prodrug compound CPF373 in phosphorylase assay:a) prodrug CPF373 under conditions of the assay in absence of the enzyme(TP) (A); b) prodrug CPF373 submitted to the action of thymidinephosphorylase (TP) (B).

COMPOUND SYNTHESIS

With reference to FIG. 1 and Schemes 1 to 3 below, compounds of thepresent invention, as exemplified by the compound CPF-373 (1), have beensynthesized using phosphorochloridate chemistry, whichphosphorochloridate chemistry has previously been reported by McGuiganet al. (1993, 1996, 1997). For example, arylphosphorodichlorophosphate(2) has been prepared coupling 1-naphthol (3) with phosphorusoxychloride (4) in the presence of Et₃N (Scheme 1) and this was allowedto react with L-alanine benzyl ester tosylate (5) in the presence ofEt₃N to generate the phosphorochloridate derivative (6) (Scheme 2). Thenucleoside 5-FdUrd (7) was converted to the 5′ ProTide by coupling withthe phosphorochloridate derivative (6) in THF, in the presence ofN-methyl imidazole (NMI) to give the target compound CPF-373 (1) (Scheme3). The sample was obtained as a mixture of two diastereoisomers asconfirmed by the presence of two peaks in the ³¹P NMR.

Anhydrous solvents were obtained from Aldrich and used without furtherpurification. All reactions were carried out under an argon atmosphere.Reactions were monitored with analytical TLC on Silica Gel 60-F254precoated aluminium plates and visualised under UV (254 nm) and/or with³¹P NMR spectra. Column chromatography was performed on silica gel(35-70 μM). Proton (¹H), carbon (¹³C), phosphorus (³¹P) and fluorine(¹⁹F) NMR spectra were recorded on a Bruker Avance 500 spectrometer at25° C. Spectra were auto-calibrated to the deuterated solvent peak andall ¹³C NMR and ³¹P NMR were proton-decoupled. Analytical HPLC wasconducted by Varian Prostar (LC Workstation-Varian prostar 335 LCdetector) using Varian Polaris C₁₈-A (10 μM) as an analytic column.

Low and High resolution mass spectra were performed as a service byBirmingham University, using electrospray (ES). CHN microanalysis wasperformed as a service by MEDAC Ltd., Surrey.

Standard Procedure A: Synthesis of Dichlorophosphate (2).

Phosphorus oxychloride (1.0 equiv) was added to a solution of 1-naphthol(1.0 equiv) in diethyl ether under argon atmosphere, then anhydroustriethylamine (1.0 equiv) was added dropwise at −78° C. and theresulting reaction mixture was stirred for 1 h. Subsequently thereaction mixture was allowed to slowly warm up to room temperature for 3h. Formation of the desired compound was monitored by ³¹P NMR. Theresulting mixture was filtered and then evaporated in vacuo undernitrogen to afford the crude colourless oil as product, which was usedwithout further purification in the next step.

Synthesis of 1-Naphthyl dichlorophosphate (2)

Prepared according to Standard Procedure A, from 1-naphthol (3.00 g,20.81 mmol), phosphorus oxychloride (1.94 mL, 20.81 mmol), triethylamine(2.9 mL, 20.81 mmol) and anhydrous diethyl ether (70 mL). After 1 h at−78° C. the reaction was left to rise to room temperature and stirredfor 3 h. The crude product was obtained as an oil. The resulting mixturewas filtered and then evaporated in vacuo, after purification by columnchromatography eluting with hexane-EtOAc, (1:1) to afford a colorlessoil (4.59 g, 84%) [R_(f)=0.93 (hexane-EtOAc, 1:1)], ³¹P NMR (202 MHz,CDCl₃): δ_(P) 5.07; ¹H NMR (500 MHz, CDCl₃): δ_(H) 7.52-7.71 (m, 4H,ArH), 7.86-7.89 (m, 1H, ArH), 7.95-7.98 (m, 1H, ArH), 8.16-8.19 (m, 1H,ArH).

Standard Procedure B: Synthesis of Phosphorochloridate (6).

A solution of aryl phosphorodichloridate (1.0 equiv.) and appropriateamino acid ester salt (1.0 equiv.) in dichloromethane under argonatmosphere was added dropwise to anhydrous triethylamine (2.0 equiv.) at−78° C. After 1 h the reaction mixture was allowed to slowly warm toroom temperature for 3 h and the formation of the desired compound wasmonitored by ³¹P NMR. The reaction mixture was concentrated underreduced pressure, the residue was redissolved in diethyl ether, filteredand evaporated in vacuo under nitrogen to afford a crude colourless oil,which in some cases was used without further purification in the nextstep. The aryl phosphorochloridate synthesized was purified by columnchromatography eluting with hexane-EtOAc, (7:3) to afford the titlecompound as a colorless oil.

Synthesis of 1-Naphthyl(benzyl-L-alaninyl) phosphorochloridate (6)

The phosphorochloridate was prepared using 1-naphthyl dichlorophosphate(2.50 g, 9.57 mmol), L-alanine benzyl ester tosylate salt (3.36 g, 9.57mmol), dry triethylamine (2.66 mL, 19.14 mmol) and dry dichloromethane(35.7 mL) according to the general procedure B. Purification by columnchromatography eluting with hexane-EtOAc, (7:3) afforded the titlecompound as a colourless oil (1.82 g, 47%) [R_(f)=0.90 (hexane-EtOAc,7:3)], ³¹P NMR (202 MHz, CDCl₃, mixture of diastereoisomers): δ_(P)7.92, 8.14 (Int.: 1.00:1.00); ¹H NMR (500 MHz, CDCl₃, mixture ofdiastereoisomers with a ratio of 1:1): δ_(H) 1.42-1.45 (m, 3H, CHCH₃),4.20-4.23 (m, 1H, CHCH₃), 4.78-4.81 (m, 1H, NH), 5.09 (s, 2H, OCH₂Ph),7.09-7.73 (m, 11H, ArH), 7.97-8.12 (m, 1H, ArH).

Standard Procedure C: Synthesis of the Nucleoside Phosphoramidate (1).

A solution of the appropriate nucleoside (1.0 equiv.) in dry THF (10 mL)was added to NMI (5.0 equiv.) at room temperature under argonatmosphere. After 10 min the reaction mixture was added dropwise to asolution of phosphorochloridate (3.0 equiv) in anhydrous THF. Thereaction was stirred at room temperature overnight and evaporated invacuo. The oil obtained was dissolved in CH₂Cl₂, washed twice with H₂O,then with HCl 0.5 M or in alternative the crude product was washed withdiethyl ether. Then the crude product was purified by columnchromatography on silica, eluting with CH₂Cl₂-MeOH as a gradient toafford the phosphoramidate.

Synthesis of 5-Fluoro-2′deoxyuridine-5′-O-[α-naphthyl(benzyl-L-alaninyl)]phosphate (1)

The phosphoramidate was prepared using 5-Fluoro-2′deoxyuridine (0.25 μg,1.01 mmol), NMI (0.40 mL, 5.07 mmol) and naphthyl(benzyl-L-alaninyl)phosphorochloridate (0.82 g, 3.04 mmol) according to the generalprocedure C. Purification by gradient column chromatography eluting withCH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded the title compound as acolourless solid (47.0 mg, 8%) [R_(f)=0.19 (CH₂Cl₂-MeOH, 95:5)], (Found:MNa⁺, 636.1520. C₂₉H₂₉N₃O₉FNaP requires [MNa⁺], 636.1523); ³¹P NMR (202MHz, MeOD, mixture of diastereoisomers): δ_(P) 4.24, 4.59; ¹⁹F NMR (470MHz, MeOD): δ_(F) −167.36, −167.18; ¹H NMR (500 MHz, MeOD): δ_(H)1.34-1.38 (m, 3H, CHCH₃), 1.67-1.79 (m, 1H, H-2′), 2.08-2.17 (m, 1H,H-2′), 4.03-4.15 (m, 2H, CHCH₃, H-4′), 4.24-4.36 (m, 3H, CH₂OP, H-3′),5.08 (d, 1H, j=12.0 Hz, OCHHPh), 5.13 (d, 1H, j=12.0 Hz, OCHHPh),6.09-6.16 (m, 1H, H-1′), 7.27-7.45 (m, 6H, ArH), 7.47-7.55 (m, 3H, ArH),7.67-7.72 (m, 2H, ArH, H-6), 7.86-7.90 (m, 1H, ArH), 8.12-8.18 (m, 1H,ArH); ¹³C NMR (125 MHz, MeOD): δ_(C) 20.3 (d, ³J_(C-P)=7.6 Hz, CH₃),20.5 (d, ³J_(C-P)=6.5 Hz, CH₃), 40.8 (CH₂), 40.9 (CH₂), 51.8 (CH), 51.9(CH), 67.6 (d, ²J_(C-P)=5.3 Hz, CH₂), 67.8 (d, ²J_(C-P)=5.2 Hz, CH₂),68.0 (CH₂), 68.1 (CH₂), 72.0 (CH), 72.1 (CH), 86.7 (d, ³J_(C-P)=8.1 Hz,CH), 86.8 (d, ³J_(C-P)=8.1 Hz, CH), 86.9 (CH), 87.0 (CH), 116.2 (d,³J_(C-P)=3.3 Hz, CH), 116.5 (d, ³J_(C-P)=3.5 Hz, CH), 122.6 (CH), 125.3(CH), 125.4 (CH), 125.6 (CH), 125.7 (CH), 126.2 (CH), 126.5 (CH), 126.6(CH), 127.6 (CH), 127.7 (CH), 127.8 (C), 127.9 (C), 128.0 (CH), 128.1(CH), 128.9 (CH), 129.0 (CH), 129.4 (CH), 129.5 (CH), 129.6 (CH), 129.7(CH), 136.2 (C), 137.1 (C), 137.2 (C), 141.6 (d, ¹J_(C-F)=233.8 Hz, C),141.7 (d, ¹J_(C-F)=233.9 Hz, C), 147.8 (d, ²J_(C-P)=7.7 Hz, C), 147.9(d, ²J_(C-P)=7.4 Hz, C), 150.5 (d, ⁴J_(C-F)=4.0 Hz, C), 159.3 (d,²J_(C-F)=26.1 Hz, C), 174.6 (d, ³J_(C-P)=5.0 Hz, C), 174.9 (d,³J_(C-P)=4.3 Hz, C), m/z (ES) 636 (MH⁺, 100%), Reverse HPLC eluting with(H₂O/MeOH from 100/0 to 0/100) in 45 min., showed two peaks of thediastereoisomers with t_(R) 34.23 min. and t_(R) 34.59 min. Anal. Calcdfor C₂₉H₂₉FN₃O₉P: C, 56.77; H, 4.76; N, 6.85. Found: C, 56.57; H, 5.06;N, 6.72.

Radioactive Pyrimidine Deoxynucleosides

[5-³H]dCyd (radiospecificity: 22 Ci/mmol) and [5-³H]dUrd(radiospecificity: 15.9 Ci/mmol) were obtained from Moravek BiochemicalsInc. (Brea, Calif.).

Standard Procedure D: Synthesis of Phosphoramidates (NMI Method)

To a stirring solution of 5-F-dUrd (1.0 eq.) in anhydrous THF, anappropriate phosphorochloridate (3.0 eq.) dissolved in anhydrous THF wasadded dropwise under an Ar atmosphere. To that reaction mixture at −78°C. was added dropwise over 5 minutes NMI (5.0 eq.). After 15 minutes,the reaction mixture was let to rise to room temperature and stirredovernight. The solvent was removed under vacuum and the residue wasre-dissolved in DCM and washed with 0.5 M HCl three times. The organiclayer was dried over MgSO₄, filtered, reduced to dryness and purified bycolumn chromatography with gradient of eluent (DCM/MeOH 99:1 to 97:3 to95:5).

Standard Procedure E: Synthesis of Phosphoramidates (tBuMgCl Method)

To a stirring solution of 5-FdUrd (1.0 eq.) dissolved in anhydrous THF,tBuMgCl (1.1 mol eq. 1M solution in THF) was added dropwise under an Aratmosphere, followed by addition (after 30 min.) of the appropriatephosphorochloridate (2.0 mol eq.) dissolved in anhydrous THF. Theresulting reaction mixture was stirred at room temperature overnight.The solvent was removed under reduced pressure and the residue waspurified by column chromatography using gradient of eluent (DCM/MeOH99:1 to 97:3 to 95:5)

5-Fluoro-2′-deoxyuridine-5′-O-[phenyl(benzoxy-L-alaninyl)]phosphate(CPF381)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.40 g,1.62 mmol), tert-butylmagnesium chloride in tetrahydrofuran (^(t)BuMgCl)(1.0 M, 2.43 mL, 2.43 mmol) and phenyl(benzoxy-L-alaninyl)phosphorochloridate (1.08 g, 3.20 mmol) according to general procedureE. Purification by gradient column chromatography on silica, elutingwith CH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded the title compound as acolourless solid (71.0 mg, 8%) [R_(f)=0.35 (CH₂Cl₂-MeOH, 95:5)], (Found:MNa⁺, 586.1360. C₂₅H₂₇N₃O₉NaPF requires [MNa⁺], 586.1367); ³¹P NMR (202MHz, MeOD): ^(TM)P 3.74, 4.14; ¹⁹F NMR (470 MHz, MeOD): ^(TM)F −167.57,−167.46; ¹H NMR (500 MHz, MeOD): ^(TM)H 1.35 (d, 3H, J=7.4 Hz, CHCH₃,one diast.), 1.37 (d, 3H, J=6.9 Hz, CHCH₃, one diast.), 1.96-2.32 (m,2H, H-2′), 3.95-4.08 (m, 2H, CHCH₃, H-4′), 4.23-4.34 (m, 3H, CH₂OP,H-3′), 5.13 (br d, 1H, =12.3 Hz, OCHHPh), 5.16 (br d, 1H, J=12.3 Hz,OCHHPh, one diast.), 5.17 (br d, 1H, J=12.2 Hz, OCHHPh, one diast.),6.16-6.22 (m, 1H, H-1′), 7.17-7.25 (m, 3H, ArH), 7.26-7.40 (m, 7H, ArH),7.81-7.85 (m, 1H, H-6); ¹³C NMR (125 MHz, MeOD): ^(TM)C 20.2 (d,³J_(C-P)=7.5 Hz, CH₃), 20.4 (d, ³J_(C-P)=6.2 Hz, CH₃), 40.6 (CH₂), 40.9(CH₂), 51.6 (CH), 51.8 (CH), 67.5 (d, ²J_(C-P)=5.3 Hz, CH₂), 67.6 (d,²J_(C-P)=5.5 Hz, CH₂), 68.0 (CH₂), 71.8 (CH), 71.9 (CH), 86.6 (d,³J_(C-P)=8.0 Hz, CH), 86.8 (d, ³J_(C-P)=8.3 Hz, CH), 86.9 (CH), 87.0(CH), 121.4 (d, ³J_(C-P)=5.1 Hz, CH), 121.5 (d, ³J_(C-P)=5.6 Hz, CH),125.5 (d, ⁵J_(C-P)=3.2 Hz, CH), 125.8 (d, ⁵J_(C-P)=3.2 Hz, CH), 126.3(CH), 129.0 (CH×2), 129.3 (CH×2), 129.6 (CH×2), 130.8 (CH×2), 140.9 (C),141.6 (d, ¹J_(C-F)=233.6 Hz, C), 141.7 (d, ¹J_(C-F)=233.6 Hz, C), 150.7(d, ⁴J_(C-F)=5.7 Hz, C), 152.1 (d, ²J_(C-F)=6.5 Hz, C), 159.2 (d,²J_(C-F)=26.3 Hz, C), 174.6 (d, ³J_(C-P)=4.9 Hz, C), 174.7 (d,³J_(C-P)=4.9 Hz, C), m/z (ES) 586 (MNa⁺, 100%); Reverse-phase HPLCeluting with H₂O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, L=275nm, showed one peak of the mixture of diastereoisomers with t_(R) 25.08min. (97%).

5-Fluoro-2′-deoxyuridine-5′-O-[phenyl(methoxy-L-alaninyl)] phosphate(CPF382) (Reference example

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g,1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmol) andphenyl(methoxy-L-alaninyl) phosphorochloridate (0.84 g, 3.04 mmol)according to general procedure D. Purification by gradient columnchromatography eluting with CH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded thetitle compound as a colourless solid (16.0 mg, 4%) [R_(f)=0.30(CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺, 510.1045. C₁₉H₂₃N₃O₉NaPF requires[MNa⁺], 510.1054); ³¹P NMR (202 MHz, MeOD): ^(TM)P 3.79, 4.09; ¹⁹F NMR(470 MHz, MeOD): ^(TM)F −167.78, −167.72; ¹H NMR (500 MHz, MeOD): ^(TM)H1.34 (d, 3H, =7.1 Hz, CHCH₃, one diast.), 1.36 (d, 3H, J=7.1 Hz, CHCH₃,one diast.), 2.02-2.16 (m, 1H, H-2′), 2.25-2.34 (m, 1H, H-2′), 3.69 (s,3H, OCH₃, one diast.), 3.70 (s, 3H, OCH₃, one diast.), 3.93-4.02 (m, 1H,CHCH₃), 4.08-4.13 (m, 1H, H-4′), 4.27-4.45 (m, 3H, CH₂OP, H-3′),6.20-6.29 (m, 1H, H-1′), 7.18-7.28 (m, 3H, ArH), 7.35-7.40 (m, 2H, ArH),7.85 (d, 1H, ³J_(H-F)=6.4 Hz, H-6); ¹³C NMR (125 MHz, MeOD): ^(TM)C 20.2(d, ³J_(C-P)=7.5 Hz, CH₃), 20.5 (d, ³J_(C-P)=6.7 Hz, CH₃), 40.8 (CH₂),40.9 (CH₂), 51.5 (CH₃), 51.6 (CH₃), 52.7 (CH), 52.8 (CH), 67.5 (d,²J_(C-P)=5.5 Hz, CH₂), 67.6 (d, ²J_(C-P)=5.1 Hz, CH₂), 72.0 (CH), 72.1(CH), 86.7 (d, ³J_(C-P)=8.2 Hz, CH), 86.8 (d, ³J_(C-P)=8.2 Hz, CH), 86.9(CH), 87.0 (CH), 121.2 (d, ³J_(C-P)=4.5 Hz, CH), 121.4 (d, ³J_(C-P)=4.7Hz, CH), 125.6 (d, ⁵J_(C-P)=2.9 Hz, CH), 125.9 (d, ⁵J_(C-P)=2.9 Hz, CH),126.2 (CH), 130.8 (CH), 130.9 (CH), 141.6 (d, ¹J_(C-F)=233.8 Hz, C),141.7 (d, ¹J_(C-F)=233.9 Hz, C), 150.6 (d, ⁴J_(C-F)=3.6 Hz, C), 152.1(d, ²J_(C-P)=6.8 Hz, C), 152.2 (d, ²J_(C-P)=6.8 Hz, C), 159.4 (d,²J_(C-F)=26.0 Hz, C), 175.2 (d, ³J_(C-P)=4.8 Hz, C), 175.5 (d,³J_(C-P)=3.7 Hz, C), m/z (ES) 510 (MNa⁺, 100%); Reverse-phase HPLCeluting with H₂O/MeOH from 100/0 to 0/100 in 45 minutes, 1

The phosphoramidate was prepared using 5-fluoro-2′deoxyuridine (0.10 g,0.40 mmol), N-methylimidazole (NMI) (0.16 mL, 2.03 mmol) andphenyl(ethoxy-L-alaninyl) phosphorochloridate (0.35 g, 1.21 mmol)according to general procedure D. Purification by gradient columnchromatography eluting with CH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded thetitle compound as a colourless solid (10.0 mg, 5%) [R_(f)=0.11(CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺, 524.1202. C₂₀H₂₅N₃O₉NaPF requires[MNa⁺], 524.1210); ³¹P NMR (202 MHz, MeOD): ^(TM)P 3.83, 4.11; ¹⁹F NMR(470 MHz, MeOD): ^(TM)F −167.67, −167.61; ¹H NMR (500 MHz, MeOD): ^(TM)H1.25 (t, 3H, =7.1 Hz, CH₂CH₃, one diast.), 1.26 (t, 3H, J=7.1 Hz,CH₂CH₃, one diast.), 1.34 (d, 3H, J=7.2 Hz, CHCH₃, one diast.), 1.36 (d,3H, J=7.2 Hz, CHCH₃, one diast.), 2.02-2.15 (m, 1H, H-2′), 2.24-2.34 (m,1H, H-2′), 3.90-4.00 (m, 1H, CHCH₃), 4.08-4.19 (m, 3H, CH₂CH₃, H-4′),4.27-4.45 (m, 3H, CH₂OP, H-3′), 6.20-6.28 (m, 1H, H-1′), 7.18-7.28 (m,3H, ArH), 7.34-7.39 (m, 2H, ArH), 7.85 (d, 1H, ³J_(H-F)=6.4 Hz, H-6);¹³C NMR (125 MHz, MeOD): ^(TM)C 14.4 (CH₃), 15.4 (CH₃), 20.3 (d,³J_(C-P)=7.6 Hz, CH₃), 20.5 (d, ³J_(C-P)=6.5 Hz, CH₃), 40.8 (CH₂), 40.9(CH₂), 51.6 (CH), 51.7 (CH), 62.4 (CH₂), 62.5 (CH₂), 67.5 (d,²J_(C-P)=5.4 Hz, CH₂), 67.6 (d, ²J_(C-P)=5.4 Hz, CH₂), 72.0 (CH), 72.1(CH), 86.7 (d, ³J_(C-P)=8.1 Hz, CH), 86.8 (d, ³J_(C-P)=8.3 Hz, CH), 86.9(CH), 87.0 (CH), 121.3 (d, ³J_(C-P)=4.8 Hz, CH), 121.4 (d, ³J_(C-P)=4.6Hz, CH), 125.6 (d, ⁵J_(C-P)=4.6 Hz, CH), 125.8 (d, ⁵J_(C-P)=4.8 Hz, CH),126.3 (CH), 130.8 (CH), 130.9 (CH), 141.6 (d, ¹J_(C-F)=233.7 Hz, C),141.8 (d, ¹J_(C-F)=233.8 Hz, C), 150.8 (br C), 152.0 (d, ²J_(C-P)=7.1Hz, C), 152.1 (d, ²J_(C-P)=7.1 Hz, C), 159.6 (d, ²J_(C-F)=26.0 Hz, C),174.8 (d, ³J_(C-P)=5.4 Hz, C), 175.1 (d, ³J_(C-P)=4.4 Hz, C), m/z (ES)524 (MNa⁺, 100%); Reverse-phase HPLC eluting with H₂O/MeOH from 100/0 to0/100 in 45 minutes, 1 ml/min, L=275 nm, showed two peaks of thediastereoisomers with t_(R) 25.63 min. and t_(R) 26.40 min. (71%:27%).

5-Fluoro-2′deoxyuridine-5′-O-[phenyl(isopropoxy-L-alaninyl)] phosphate(CPF384)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g,1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmol) andphenyl(isopropoxy-L-alaninyl) phosphorochloridate (0.93 g, 3.04 mmol)according to general procedure D. Purification by gradient columnchromatography eluting with CH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded thetitle compound as a colourless solid (31.0 mg, 6%) [R_(f)=0.21(CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺, 538.1370. C₂₁H₂₇N₃O₉NaPF requires[MNa⁺], 538.1367); ³¹P NMR (202 MHz, MeOD): ^(TM)P 3.87, 4.13; ¹⁹F NMR(470 MHz, MeOD): ^(TM)F −167.64, −167.56; ¹H NMR (500 MHz, MeOD): ^(TM)H1.22-1.26 (m, 6H, CH(CH₃)₂), 1.33 (d, 3H, J=7.1 Hz, CHCH₃, one diast.),1.35 (d, 3H, J=7.1 Hz, CHCH₃, one diast.), 2.00-2.15 (m, 1H, H-2′),2.23-2.34 (m, 1H, H-2′), 3.88-3.96 (m, 1H, CHCH₃), 4.08-4.14 (m, 1H,H-4′), 4.27-4.45 (m, 3H, CH₂OP, H-3′), 4.98 (hept, 1H, j=6.1 Hz,CH(CH₃)₂), 6.20-6.29 (m, 1H, H-1′), 7.17-7.29 (m, 3H, Ar—H), 7.34-7.40(m, 2H, Ar—H), 7.84 (d, 1H, ³J_(H-F)=6.4 Hz, H-6); ¹³C NMR (125 MHz,MeOD): ^(TM)C 20.3 (d, ³J_(C-P)=7.6 Hz, CH₃), 20.5 (d, ³J_(C-P)=6.4 Hz,CH₃), 21.9 (CH₃×2), 22.0 (CH₃×2), 40.8 (CH₂), 40.9 (CH₂), 51.7 (CH),51.8 (CH), 67.5 (d, 2J_(C-P)=5.4 Hz, CH₂), 67.6 (d, 2J_(C-P)=5.2 Hz,CH₂), 70.2 (CH), 70.3 (CH), 72.0 (CH), 72.1 (CH), 86.6 (d, ³J_(C-P)=8.2Hz, CH), 86.8 (d, ³J_(C-P)=8.2 Hz, CH), 86.9 (CH), 87.0 (CH), 121.2 (d,³J_(C-P)=4.7 Hz, CH), 121.4 (d, ³J_(C-P)=4.9 Hz, CH), 125.6 (d,⁵J_(C-P)=7.1 Hz, CH), 125.9 (d, ⁵J_(C-P)=7.1 Hz, CH), 126.3 (CH), 130.8(CH), 130.9 (CH), 141.8 (d, ¹J_(C-F)=234.5 Hz, C), 141.9 (d,¹J_(C-F)=234.4 Hz, C), 150.7 (d, ⁴J_(C-F)=3.7 Hz, C), 152.0 (d,³J_(C-P)=6.2 Hz, C), 152.1 (d, ³J_(C-P)=6.2 Hz, C), 159.3 (d,²J_(C-F)=26.3 Hz, C), 159.4 (d, ²J_(C-F)=26.0 Hz, C), 174.3 (d,³J_(C-P)=5.6 Hz, C), 174.6 (d, ³J_(C-P)=4.6 Hz, C), m/z (ES) 538 (MNa⁺,100%); Reverse-phase HPLC eluting with H₂O/MeOH from 100/0 to 0/100 in45 minutes, 1 ml/min, L=275 nm, showed two peaks of the diastereoisomerswith t_(R) 28.93 min. and t_(R) 29.45 min. (44%: 52%).

5-Fluoro-2′deoxyuridine-5′-O-[phenyl (cyclohexoxy-L-alaninyl)] phosphate(CPF508

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridne (0.30 g,1.21 mmol), N-methylimidazole (NMI) (0.48 mL, 6.09 mmol) andphenyl(cyclohexoxy-L-alaninyl) phosphorochloridate (1.026 g, 3.65 mmol)according to general procedure D. Purification by gradient columnchromatography eluting with CH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded thetitle compound as a colourless solid (6.7 mg, 3%) [R_(f)=0.45(CH₂Cl₂-MeOH, 95:5)]; (Found: MNa⁺, 565.48. C₂₄H₃₁N₃O₉NaPF requires[MNa⁺], 565.49); ³¹P NMR (202 MHz, MeOD): ^(TM)P 3.86, 4.15; ¹⁹F NMR(470 MHz, MeOD): ^(TM)F −167.68, −167.62; ¹H NMR (500 MHz, MeOD): ^(TM)H1.26-1.40 (m, 3H, CHCH₃), 1.41-1.50 (m, 4H, CH(CH₂)₅), 1.52-1.61 (m, 1H,CH(CH₂)₅), 1.70-1.88 (m, 5H, CH(CH₂)₅), 2.00-2.14 (m, 1H, H-2′),2.23-2.34 (m, 1H, H-2′), 3.90-3.98 (m, 1H, CHCH₃), 4.07-4.14 (m, 1H,H-4′), 4.29-4.39 (m, 2H, CH₂OP), 4.40-4.45 (m, 1H, H-3′), 4.72-4.78 (m,1H, CH(CH₂)s), 6.20-6.28 (m, 1H, H-1′), 7.18-7.29 (m, 3H, ArH),7.34-7.39 (m, 2H, ArH), 7.85 (d, 1H, ³J_(H-F)=6.6 Hz, H-6); ¹³C NMR (125MHz, MeOD): ^(TM)C 20.3 (d, ³J_(C-P)=7.3 Hz, CH₃), 20.6 (d, ³J_(C-P)=6.5Hz, CH₃), 24.6 (CH₂), 26.4 (CH₂), 32.3 (CH₂), 32.4 (CH₂), 40.9 (CH₂),51.7 (CH), 51.9 (CH), 67.5 (d, 2J_(C-P)=5.3 Hz, CH₂), 67.7 (d,²J_(C-P)=5.3 Hz, CH₂), 72.0 (CH), 72.1 (CH), 74.9 (CH), 86.6 (d,³J_(C-P)=8.5 Hz, CH), 86.8 (d, ³J_(C-P)=8.5 Hz, CH), 86.9 (CH), 87.0(CH), 121.3 (CH), 121.4 (CH), 121.5 (CH), 121.6 (CH), 125.6 (CH), 125.7(CH), 125.8 (CH), 125.9 (CH), 126.3 (CH), 130.1 (CH), 141.5 (d,¹J_(C-F)=234.0 Hz, C), 150.7 (d, ⁴J_(C-P)=4.0 Hz, C), 152.0 (d,²J_(C-P)=7.2 Hz, C), 152.1 (d, ²J_(C-P)=7.2 Hz, C), 159.4 (d,²J_(C-F)=26.3 Hz, C), 174.3 (d, ³J_(C-P)=4.6 Hz, C), 174.5 (d,³J_(C-P)=4.3 Hz, C); m/z (ES) 565 (MNa⁺, 100%); Reverse-phase HPLCeluting with H₂O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, L=275nm, showed two peaks of the diastereoisomers with t_(R) 30.00 min. andt_(R) 30.45 min. (33%: 65%).

5-Fluoro-2′deoxyuridine-5′-O-[p-nitro-phenyl(ethoxy-L-alaninyl)]phosphate (CPF430)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g,1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmol) andp-nitro-phenyl(ethoxy-L-alaninyl) phosphorochloridate (1.02 g, 3.04mmol) according to general procedure D. Purification by gradient columnchromatography eluting with CH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded thetitle compound as a colourless solid (77.0 mg, 14%) [R_(f)=0.24(CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺, 569.1066. C₂₀H₂₄N₄O₁₁NaPF requires[MNa⁺], 569.1061); ³¹P NMR (202 MHz, MeOD): ^(TM)P 3.63, 3.67; ¹⁹F NMR(470 MHz, MeOD): ^(TM)F −167.89, −167.82; ¹H NMR (500 MHz, MeOD): ^(TM)H1.24 (t, 3H, J=7.0 Hz, CH₂CH₃), 1.25 (t, 3H, j=7.0 Hz, CH₂CH₃),1.36-1.40 (m, 3H, CHCH₃), 2.16-2.25 (m, 1H, H-2′), 2.30-2.38 (m, 1H,H-2′), 3.95-4.00 (m, 1H, CHCH₃), 4.09-4.19 (m, 3H, CH₂CH₃, H-4′),4.32-4.48 (m, 3H, CH₂OP, H-3′), 6.21-6.29 (m, 1H, H-1′), 7.46 (d, 1H,j=8.7 Hz, ArH), 7.49 (d, 1H, j=8.7 Hz, ArH), 7.85 (d, 1H, ³J_(H-F)=6.6Hz, H-6), 7.87 (d, 1H, ³J_(H-F)=6.6 Hz, H-6), 8.29 (d, 2H, =8.7 Hz,ArH); ¹³C NMR (125 MHz, MeOD): ^(TM)C 14.5 (CH₃), 14.6 (CH₃), 20.3 (d,³J_(C-P)=7.5 Hz, CH₃), 20.4 (d, ³J_(C-P)=6.4 Hz, CH₃), 40.8 (CH₂), 51.6(CH), 51.7 (CH), 62.5 (CH₂), 67.8 (d, 2J_(C-P)=5.5 Hz, CH₂), 68.0 (d,2J_(C-P)=5.2 Hz, CH₂), 71.8 (CH×2), 86.4 (CH), 86.5 (CH), 87.0 (d,³J_(C-P)=7.5 Hz, CH), 122.1 (d, ³J_(C-P)=5.2 Hz, CH), 122.5 (d,³J_(C-P)=5.0 Hz, CH), 125.7 (CH), 126.0 (CH), 126.6 (CH), 141.3 (d,¹J_(C-F)=233.6 Hz, C), 141.5 (d, ¹J_(C-F)=233.7 Hz, C), 146.2 (C), 150.6(d, ⁴J_(C-P)=4.6 Hz, C), 156.9 (d, ²J_(C-P)=2.6 Hz, C), 157.0 (d,²J_(C-P)=2.6 Hz, C), 159.3 (d, ²J_(C-F)=26.3 Hz, C), 174.6 (d,³J_(C-P)=4.6 Hz, C), 174.9 (d, ³J_(C-P)=3.7 Hz, C), m/z (ES) 569 (MNa⁺,100%); Reverse-phase HPLC eluting with H₂O/MeOH from 100/0 to 0/100 in45 min., 1 ml/min, L=275 nm, showed two peaks of the diastereoisomerswith t_(R) 31.63 min. and t_(R) 31.89 min. (11%: 85%).

5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (benzoxy-L-alaninyl)] phosphate(CPF373)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g,1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmol) and1-naphthyl(benzoxy-L-alaninyl) phosphorochloridate (0.82 g, 3.04 mmol)according to general procedure D. Purification by gradient columnchromatography eluting with CH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded thetitle compound as a colourless solid (47.0 mg, 8%) [R_(f)=0.19(CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺, 636.1520. C₂₉H₂₉N₃O₉NaPF requires[MNa⁺], 636.1523); ³¹P NMR (202 MHz, MeOD): ^(TM)P 4.24, 4.59; ¹⁹F NMR(470 MHz, MeOD): ^(TM)F −167.36, −167.18; ¹H NMR (500 MHz, MeOD): ^(TM)H1.34-1.38 (m, 3H, CHCH₃), 1.67-1.79 (m, 1H, H-2′), 2.08-2.17 (m, 1H,H-2′), 4.03-4.15 (m, 2H, CHCH₃, H-4′), 4.24-4.36 (m, 3H, CH₂OP, H-3′),5.08 (d, 1H, J=12.0 Hz, OCHHPh), 5.13 (d, 1H, J=12.0 Hz, OCHHPh),6.09-6.16 (m, 1H, H-1′), 7.27-7.45 (m, 6H, ArH), 7.47-7.55 (m, 3H, ArH),7.67-7.72 (m, 2H, ArH, H-6), 7.86-7.90 (m, 1H, ArH), 8.12-8.18 (m, 1H,ArH); ¹³C NMR (125 MHz, MeOD): ^(TM)C 20.3 (d, ³J_(C-P)=7.6 Hz, CH₃),20.5 (d, ³J_(C-P)=6.5 Hz, CH₃), 40.8 (CH₂), 40.9 (CH₂), 51.8 (CH), 51.9(CH), 67.6 (d, ²J_(C-P)=5.3 Hz, CH₂), 67.8 (d, ²J_(C-P)=5.2 Hz, CH₂),68.0 (CH₂), 68.1 (CH₂), 72.0 (CH), 72.1 (CH), 86.7 (d, ³J_(C-P)=8.1 Hz,CH), 86.8 (d, ³J_(C-P)=8.1 Hz, CH), 86.9 (CH), 87.0 (CH), 116.2 (d,³J_(C-P)=3.3 Hz, CH), 116.5 (d, ³J_(C-P)=3.5 Hz, CH), 122.6 (CH), 125.3(CH), 125.4 (CH), 125.6 (CH), 125.7 (CH), 126.2 (CH), 126.5 (CH), 126.6(CH), 127.6 (CH), 127.7 (CH), 127.8 (C), 127.9 (C), 128.0 (CH), 128.1(CH), 128.9 (CH), 129.0 (CH), 129.4 (CH), 129.5 (CH), 129.6 (CH), 129.7(CH), 136.2 (C), 137.1 (C), 137.2 (C), 141.6 (d, ¹J_(C-F)=233.8 Hz, C),141.7 (d, ¹J_(C-F)=233.9 Hz, C), 147.8 (d, ²J_(C-P)=7.7 Hz, C), 147.9(d, ²J_(C-P)=7.4 Hz, C), 150.5 (d, ⁴J_(C-F)=4.0 Hz, C), 159.3 (d,²J_(C-F)=26.1 Hz, C), 174.6 (d, ³J_(C-P)=5.0 Hz, C), 174.9 (d,³J_(C-P)=4.3 Hz, C), m/z (ES) 636 (MNa⁺, 100%); Reverse-phase HPLCeluting with H₂O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, L=275nm, showed two peaks of the diastereoisomers with t_(R) 34.23 min. andt_(R) 34.59 min. (23%: 76%).

5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (methoxy-L-alaninyl)] phosphate(CPF385)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g,1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmol) and1-naphthyl(methoxy-L-alaninyl) phosphorochloridate (0.99 g, 3.04 mmol)according to general procedure D. Purification by gradient columnchromatography eluting with CH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded thetitle compound as a colourless solid (7.0 mg, 1%) [R_(f)=0.23(CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺, 560.1198. C₂₃H₂₅N₃O₉NaPF requires[MNa⁺], 560.1210); ³¹P NMR (202 MHz, MeOD): ^(TM)P 4.31, 4.56; ¹⁹F NMR(470 MHz, MeOD): ^(TM)F −167.51, −167.37; ¹H NMR (500 MHz, MeOD): ^(TM)H1.34 (d, 3H, J=6.7 Hz, CHCH₃, one diast.), 1.36 (d, 3H, J=6.7 Hz, CHCH₃,one diast.), 1.76-1.87 (m, 1H, H-2′), 2.12-2.22 (m, 1H, H-2′), 3.64 (s,3H, OCH₃, one diast.), 3.65 (s, 3H, OCH₃, one diast.), 4.03-4.13 (m, 2H,CHCH₃, H-4′), 4.30-4.38 (m, 2H, CH₂OP), 4.41 (dd, 1H, J=2.5 Hz, J=5.8Hz, H-3′), 6.12-6.19 (m, 1H, H-1′), 7.41-7.46 (m, 1H, ArH), 7.50-7.58(m, 3H, ArH), 7.70-7.76 (m, 2H, H-6, ArH), 7.87-7.91 (m, 1H, ArH),8.15-8.20 (m, 1H, ArH); ¹³C NMR (125 MHz, MeOD): ^(TM)C 20.3 (d,³J_(C-P)=7.1 Hz, CH₃), 20.4 (d, ³J_(C-P)=6.5 Hz, CH₃), 40.7 (CH₂), 40.8(CH₂), 51.6 (CH₃), 51.7 (CH₃), 52.7 (CH), 52.8 (CH), 67.8 (d,²J_(C-P)=5.7 Hz, CH₂), 67.5 (d, ²J_(C-P)=5.7 Hz, CH₂), 72.0 (CH), 72.1(CH), 86.7 (d, ³J_(C-P)=7.9 Hz, CH), 86.9 (d, ³J_(C-P)=8.5 Hz, CH), 86.9(CH), 87.0 (CH), 116.2 (d, ³J_(C-P)=3.1 Hz, CH), 116.5 (d, ³J_(C-P)=3.5Hz, CH), 122.5 (CH), 122.6 (CH), 125.4 (CH), 125.5 (CH), 125.6 (CH),125.7 (CH), 126.1 (CH), 126.2 (CH), 126.5 (CH), 126.6 (CH), 127.6 (CH),127.7 (C×2), 127.8 (CH), 127.9 (CH), 128.9 (CH), 129.0 (CH), 136.3 (C),141.6 (d, ¹J_(C-F)=233.4 Hz, C), 141.7 (d, ¹J_(C-F)=234.1 Hz, C), 147.8(d, ²J_(C-P)=7.9 Hz, C), 148.0 (d, ²J_(C-P)=7.2 Hz, C), 150.6 (C), 159.4(d, ²J_(C-F)=27.0 Hz, C), 175.2 (d, ³J_(C-P)=3.9 Hz, C), 175.5 (d,³J_(C-P)=3.9 Hz, C), m/z (ES) 560 (MNa⁺, 100%); Reverse-phase HPLCeluting with H₂O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, L=275nm, showed two peaks of the diastereoisomers with t_(R) 28.45 min. andt_(R) 28.85 min. (73%: 25%).

5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (ethoxy-L-alaninyl)] phosphate(CPF386)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.25 g,1.01 mmol), N-methylimidazole (NMI) (0.40 mL, 5.07 mmol) and1-naphthyl(ethoxy-L-alaninyl) phosphorochloridate (1.04 g, 3.04 mmol)according to general procedure D. Purification by gradient columnchromatography eluting with CH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded thetitle compound as a colourless solid (47.0 mg, 4%) [R_(f)=0.25(CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺, 574.1360. C₂₄H₂₇N₃O₉NaPF requires[MNa⁺], 574.1367); ³¹P NMR (202 MHz, MeOD): ^(TM)P 4.34, 4.55; ¹⁹F NMR(470 MHz, MeOD): ^(TM)F −167.31, −167.16; ¹H NMR (500 MHz, MeOD): ^(TM)H1.20 (t, 3H, =7.0 Hz, CH₂CH₃, one diast.), 1.21 (t, 3H, =7.0 Hz, CH₂CH₃,one diast.), 1.33-1.37 (m, 3H, CHCH₃), 1.73-1.86 (m, 1H, H-2′),2.12-2.21 (m, 1H, H-2′), 4.01-4.07 (m, 1H, CHCH₃), 4.08-4.13 (m, 3H,CH₂CH₃, H-4′), 4.31-4.43 (m, 3H, CH₂OP, H-3′), 6.11-6.19 (m, 1H, H-1′),7.39-7.46 (m, 1H, ArH), 7.50-7.57 (m, 3H, ArH), 7.68-7.75 (m, 2H, ArH,H-6), 7.86-7.91 (m, 1H, ArH), 8.15-8.20 (m, 1H, ArH); ¹³C NMR (125 MHz,MeOD): ^(TM)c 14.4 (CH₃), 20.3 (d, ³J_(C-P)=7.4 Hz, CH₃), 20.5 (d,³J_(C-P)=6.2 Hz, CH₃), 40.8 (CH₂), 40.9 (CH₂), 51.8 (CH), 51.9 (CH),62.4 (CH₂), 62.5 (CH₂), 67.8 (d, ²J_(C-P)=4.6 Hz, CH₂), 67.9 (d,²J_(C-P)=4.6 Hz, CH₂), 72.0 (CH), 72.1 (CH), 86.7 (d, ³J_(C-P)=8.4 Hz,CH), 86.8 (d, ³J_(C-P)=8.4 Hz, CH), 86.9 (CH), 87.0 (CH), 116.1 (d,³J_(C-P)=3.5 Hz, CH), 116.5 (d, ³J_(C-P)=3.5 Hz, CH), 122.6 (CH), 125.4(CH), 125.5 (CH), 125.7 (CH), 125.8 (CH), 126.1 (CH), 126.2 (CH), 126.5(CH), 126.6 (CH), 127.5 (CH), 127.6 (C), 127.7 (C), 127.8 (CH), 127.9(CH), 128.9 (CH), 129.0 (CH), 136.3 (C), 141.6 (d, ¹J_(C-F)=233.3 Hz,C), 141.7 (d, ¹J_(C-F)=233.4 Hz, C), 147.8 (d, ²J_(C-P)=6.9 Hz, C),148.0 (d, ²J_(C-P)=6.9 Hz, C), 150.6 (C), 159.3 (d, ²J_(C-F)=26.3 Hz,C), 174.8 (d, ³J_(C-P)=4.8 Hz, C), 175.1 (d, ³J_(C-P)=4.0 Hz, C); m/z(ES) 574 (MNa⁺, 100%); Reverse-phase HPLC eluting with H₂O/MeOH from100/0 to 0/100 in 45 minutes, 1 ml/min, L=275 nm, showed two peaks ofthe diastereoisomers with t_(R) 30.77 min. and t_(R) 31.20 min. (51%:48%).

5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl(isopropoxy-L-alaninyl)]phosphate (CPF387)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.10 g,0.40 mmol), tert-butylmagnesium chloride in tetrahydrofuran (^(t)BuMgCl)(1.0 M, 0.61 mL, 0.61 mmol) and 1-naphthyl(isopropoxy-L-alaninyl)phosphorochloridate (0.31 g, 0.89 mmol) according to general procedureE. Purification by gradient column chromatography eluting with CH₂Cl₂until CH₂Cl₂-MeOH (95:5) afforded the title compound as a colourlesssolid (71.0 mg, 17%) [R_(f)=0.21 (CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺,588.1521. C₂₅H₂₉N₃O₉NaPF requires [MNa⁺], 588.1523); ³¹P NMR (202 MHz,MeOD): ^(TM)P 4.38, 4.58; ¹⁹F NMR (470 MHz, MeOD): ^(TM)F −167.43,−167.26; ¹H NMR (500 MHz, MeOD): ^(TM)H 1.19-1.23 (m, 6H, CH(CH₃)₂),1.34-1.38 (m, 3H, CHCH₃), 1.68-1.84 (m, 1H, H-2′), 2.09-2.20 (m, 1H,H-2′), 3.96-4.05 (m, 1H, CHCH₃), 4.07-4.12 (m, 1H, H-4′), 4.29-4.38 (m,2H, CH₂OP), 4.39-4.42 (m, 1H, H-3′), 4.93-5.01 (m, 1H, CH(CH₃)₂),5.10-6.18 (m, 1H, H-1′), 7.40-7.46 (m, 1H, ArH), 7.50-7.57 (m, 3H, ArH),7.70-7.75 (m, 2H, H-6, ArH), 7.87-7.92 (m, 1H, ArH), 8.16-8.20 (m, 1H,ArH); ¹³C NMR (125 MHz, MeOD): ^(TM)C 20.3 (d, ³J_(C-P)=7.1 Hz, CH₃),20.5 (d, ³J_(C-P)=6.6 Hz, CH₃), 21.8 (CH₃), 21.9 (CH₃), 22.0 (CH₃), 22.1(CH₃), 40.8 (CH₂), 40.9 (CH₂), 51.9 (CH), 52.0 (CH), 67.8 (d,²J_(C-P)=4.5 Hz, CH₂), 67.9 (d, ²J_(C-P)=4.8 Hz, CH₂), 70.2 (CH), 70.3(CH), 72.0 (CH), 72.1 (CH), 86.6 (CH), 86.7 (CH), 86.9 (d, ³J_(C-P)=8.6Hz, CH), 87.0 (d, ³J_(C-P)=8.6 Hz, CH), 116.2 (d, ³J_(C-P)=2.5 Hz, CH),116.5 (d, ³J_(C-P)=2.7 Hz, CH), 122.6 (CH), 125.5 (CH), 125.7 (CH),126.1 (CH), 126.2 (CH), 126.5 (CH), 127.5 (CH), 127.6 (C), 127.7 (C),127.8 (CH), 127.9 (CH), 128.9 (CH), 129.0 (CH), 136.3 (C), 141.6 (d,¹J_(C-F)=233.2 Hz, C), 141.7 (d, ¹J_(C-F)=233.4 Hz, C), 147.7 (d,²J_(C-P)=7.6 Hz, C), 147.9 (d, ²J_(C-P)=7.7 Hz, C), 150.5 (C), 159.4 (d,²J_(C-F)=26.2 Hz, C), 174.4 (d, ³J_(C-P)=5.0 Hz, C), 174.7 (d,³J_(C-P)=5.1 Hz, C); m/z (ES) 588 (MNa⁺, 100%); Reverse-phase HPLCeluting with H₂O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, L=275nm, showed two peaks of the diastereoisomers with t_(R) 32.20 min. andt_(R) 32.80 min. (27%: 69%).

5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl(cyclohexoxy-L-alaninyl)]phosphate (CPF509)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.30 g,1.21 mmol), N-methylimidazole (NMI) (0.48 mL, 6.09 mmol) andphenyl(cyclohexoxy-L-alaninyl) phosphorochloridate (1.45 g, 3.65 mmol)according to general procedure D. Purification by gradient columnchromatography eluting with CH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded thetitle compound as a colourless solid (6.7 mg, 3%) [R_(f)=0.47(CH₂Cl₂-MeOH, 95:5)]; (Found: MNH₄ ⁺, 623.2261. C₂₈H₃₇N₄O₉NaPF requires[MNH₄+], 623.2282); ³¹P NMR (202 MHz, MeOD): ^(TM)P 4.35, 4.52; ¹⁹F NMR(470 MHz, MeOD): ^(TM)F −167.31, −167.17; ¹H NMR (500 MHz, MeOD): ^(TM)H1.30-1.43 (m, 3H, CHCH₃), 1.44-1.56 (m, 4H, CH(CH₂)₅), 1.57-1.66 (m, 1H,CH(CH₂)₅), 1.67-1.83 (m, 5H, CH(CH₂)₅), 1.84-1.93 (m, 1H, H-2′),2.09-2.20 (m, 1H, H-2′), 3.98-4.06 (m, 1H, CHCH₃), 4.07-4.15 (m, 1H,H-4′), 4.29-4.38 (m, 2H, CH₂OP), 4.39-4.44 (m, 1H, H-3′), 4.67-4.76 (m,1H, CH(CH₂)₅), 6.09-6.19 (m, 1H, H-1′), 7.38-7.57 (m, 5H, ArH),7.68-7.75 (m, 1H, ArH), 7.79-7.92 (m, 1H, ArH), 8.17 (d, 1H,³J_(H-F)=6.6 Hz, H-6); ¹³C NMR (125 MHz, MeOD): ^(TM)C 20.4 (d,³J_(C-P)=8.0 Hz, CH₃), 20.6 (d, ³J_(C-P)=6.5 Hz, CH₃), 24.5 (CH₂), 26.3(CH₂), 32.3 (CH₂), 40.8 (CH₂), 51.8 (CH), 51.9 (CH), 67.8 (CH₂), 72.0(CH), 72.2 (CH), 75.0 (CH), 86.7 (d, ³J_(C-P)=8.2 Hz, CH), 87.0 (CH),116.1 (d, ³J_(C-P)=2.5 Hz, CH), 116.4 (d, ³J_(C-P)=3.0 Hz, CH), 122.6(CH), 124.8 (CH), 125.9 (CH), 126.1 (CH), 126.2 (CH), 126.4 (CH), 126.5(CH), 126.6 (CH), 127.6 (CH), 127.7 (C×2), 127.8 (CH), 127.9 (CH), 128.9(CH), 129.0 (CH), 136.3 (C), 141.6 (C), 148.0 (d, ²J_(C-P)=7.2 Hz, C),150.6 (C), 159.4 (d, ²J_(C-F)=27.0 Hz, C), 175.2 (d, ³J_(C-P)=3.9 Hz,C), 175.5 (d, ³J_(C-P)=3.9 Hz, C); m/z (ES) 623 (MNH₄ ⁺, 100%);Reverse-phase HPLC eluting with H₂O/MeOH from 100/0 to 0/100 in 45minutes, 1 ml/min, L=275 nm, showed two peaks of the diastereoisomerswith t_(R) 30.50 min. and t_(R) 31.48 min. (27%: 69%).

5-Fluoro-2′deoxyuridine-5′-O-[phenyl(benzoxy-α,α-dimethylglycine)]phosphate (CPF393)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.40 g,1.62 mmol), tert-butylmagnesium chloride in tetrahydrofuran (^(t)BuMgCl)(1.0 M, 2.43 mL, 2.43 mmol) and phenyl(benzoxy-α,α-dimethylglycine)phosphorochloridate (1.17 g, 3.20 mmol) according to general procedureE. Purification by gradient column chromatography eluting with CH₂Cl₂until CH₂Cl₂-MeOH (95:5) afforded the title compound as a colourlesssolid (69.0 mg, 7%) [R_(f)=0.27 (CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺,600.1527. C₂₆H₂₉N₃O₉NaPF requires [MNa⁺], 600.1523); ³¹P NMR (202 MHz,MeOD): ^(TM)P 2.42, 2.47; ¹⁹F NMR (470 MHz, MeOD): ^(TM)F −167.80,−167.62; ¹H NMR (500 MHz, MeOD): ^(TM)H 1.51-1.60 (m, 6H, C(CH₃)₂),1.89-1.97 (m, 1H, H-2′, one diast.), 2.07-2.15 (m, 1H, H-2′, onediast.), 2.21 (ddd, 1H, J=3.4 Hz, 5.9 Hz, 13.5 Hz, H-2′, one diast.),2.29 (ddd, 1H, J=3.2 Hz, 6.1 Hz, 13.5 Hz, H-2′, one diast.), 4.00-4.07(m, 1H, H-4′), 4.22-4.31 (m, 2H, CH₂OP), 4.32-4.36 (m, 1H, H-3′, onediast.), 4.37-4.41 (m, 1H, H-3′, one diast.), 5.08-5.18 (m, 2H, OCH₂Ph),6.19-6.25 (m, 1H, H-1′), 7.20-7.26 (m, 3H, ArH), 7.27-7.39 (m, 7H, ArH),7.74 (d, ³J_(H-F)=6.4 Hz, H-6, one diast.), 7.80 (d, ³J_(H-F)=6.4 Hz,H-6, one diast.); ¹³C NMR (125 MHz, MeOD): ^(TM)C 27.5 (CH₃), 27.7 (d,³J_(C-P)=7.1 Hz, CH₃), 27.8 (d, ³J_(C-P)=7.1 Hz, CH₃), 40.8 (CH₂), 40.9(CH₂), 58.2 (C), 58.3 (C), 67.6 (d, 2J_(C-P)=5.5 Hz, CH₂), 67.7 (d,2J_(C-P)=5.5 Hz, CH₂), 68.3 (CH₂), 71.9 (CH), 72.0 (CH), 86.6 (d,³J_(C-P)=8.1 Hz, CH), 86.8 (d, ³J_(C-P)=7.3 Hz, CH), 86.9 (CH), 121.4(d, ³J_(C-P)=4.8 Hz, CH), 121.6 (d, ³J_(C-P)=4.5 Hz, CH), 125.6 (CH),125.8 (CH), 125.9 (CH), 126.1 (CH), 126.2 (CH), 129.3 (CH), 129.4 (CH),129.6 (CH), 130.7 (CH), 130.8 (CH), 137.2 (C), 137.3 (C), 141.8 (d,¹J_(C-F)=233.7 Hz, C), 150.6 (C), 152.1 (d, ⁴J_(C-F)=7.0 Hz, C), 152.1(d, ⁴J_(C-F)=7.6 Hz, C), 159.3 (d, ²J_(C-F)=26.1 Hz, C), 159.4 (d,2J_(C-F)=26.1 Hz, C), 176.5 (d, ³J_(C-P)=4.0 Hz, C), 176.6 (d,³J_(C-P)=3.8 Hz, C), m/z (ES) 600.1 (MNa⁺, 100%); Reverse-phase HPLCeluting with H₂O/MeOH from 100/0 to 0/100 in 35 minutes, 1 ml/min, L=275nm, showed one peak of the mixture of diastereoisomers with t_(R) 17.71(96%).

5-Fluoro-2′deoxyuridine-5′-O-[phenyl(ethoxy-α,α-dimethylglycine)]phosphate (CPF394)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.20 g,0.80 mmol), N-methylimidazole (NMI) (0.31 mL, 4.0 mmol) andphenyl(ethoxy-α,α-dimethylglycine) phosphorochloridate (0.73 g, 2.40mmol) according to general procedure D. Purification by gradient columnchromatography eluting with CH₂Cl₂ until CH₂Cl₂-MeOH (95:5) afforded thetitle compound as a colourless solid (25.0 mg, 6%) [R_(f)=0.24(CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺, 538.1367. C₂₁H₂₇N₃O₉NaPF requires[MNa⁺], 538.1367); ³¹P NMR (202 MHz, MeOD): ^(TM)P 2.49, 2.52; ¹⁹F NMR(470 MHz, MeOD): ^(TM)F −167.62, −167.58; ¹H NMR (500 MHz, MeOD): ^(TM)H1.24 (t, 3H, =7.1 Hz, CH₂CH₃, one diast.), 1.26 (t, 3H, j=7.1 Hz,CH₂CH₃, one diast.), 1.44-1.54 (m, 6H, C(CH₃)₂), 1.95-2.04 (m, 1H, H-2′,one diast.), 2.13-2.21 (m, 1H, H-2′, one diast.), 2.24 (ddd, 1H, j=3.1Hz, =6.3 Hz, =13.5 Hz, H-2′, one diast.), 2.31 (ddd, 1H, =3.2 Hz, =6.1Hz, =13.7 Hz, H-2′, one diast.), 4.08-4.19 (m, 3H, CH₂CH₃, H-4′),4.33-4.49 (m, 3H, CH₂OP, H-3′), 6.20-6.30 (m, 1H, H-1′), 7.23-7.28 (m,3H, ArH), 7.33-7.40 (m, 2H, ArH), 7.80 (d, ³J_(H-F)=6.4 Hz, H-6, onediast.), 7.88 (d, ³J_(H-F)=6.4 Hz, H-6, one diast.); ¹³C NMR (125 MHz,MeOD): ^(TM)C 14.4 (CH₃), 14.5 (CH₃), 27.5 (d, ³J_(C-P)=7.3 Hz, CH₃),27.7 (d, ³J_(C-P)=7.6 Hz, CH₃), 27.8 (d, ³J_(C-P)=7.6 Hz, CH₃), 40.8(CH₂), 40.9 (CH₂), 58.1 (C), 62.6 (CH₂), 62.7 (CH₂), 67.6 (d,²J_(C-P)=6.7 Hz, CH₂), 67.7 (d, ²J_(C-P)=5.8 Hz, CH₂), 71.9 (CH), 72.0(CH), 86.6 (d, ³J_(C-P)=8.1 Hz, CH), 86.8 (d, ³J_(C-P)=7.6 Hz, CH), 86.9(CH), 121.4 (d, ³J_(C-P)=4.4 Hz, CH), 121.6 (d, ³J_(C-P)=4.4 Hz, CH),125.6 (CH), 125.8 (CH), 125.9 (CH), 126.1 (CH), 126.2 (CH), 130.7 (CH),130.8 (CH), 130.9 (CH), 141.8 (d, ¹J_(C-F)=233.5 Hz, C), 150.6 (C),150.7 (C), 152.2 (d, ⁴J_(C-F)=7.3 Hz, C), 152.3 (d, ⁴J_(C-F)=6.9 Hz, C),159.2 (d, ²J_(C-F)=20.3 Hz, C), 159.4 (d, ²J_(C-F)=20.4 Hz, C), 176.6(d, ³J_(C-P)=4.2 Hz, C), 176.8 (d, ³J_(C-P)=4.6 Hz, C), m/z (ES) 538.1(MNa⁺, 100%); Reverse-phase HPLC eluting with H₂O/MeOH from 100/0 to0/100 in 45 minutes, 1 ml/min, L=275 nm, showed two peaks of thediastereoisomers with t_(R) 18.76 min. and t_(R) 20.44 min. (68%: 30%).

5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl(benzoxy-α,α-dimethylglycine)]phosphate (CPF395)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.40 g,1.62 mmol), N-methylimidazole (NMI) (0.64 mL, 8.0 mmol) and1-naphthyl(benzoxy-α,α-dimethylglycine) phosphorochloridate (2.00 g,4.80 mmol) according to general procedure D. Purification by gradientcolumn chromatography eluting with CH₂Cl₂ until CH₂Cl₂-MeOH (95:5)afforded the title compound as a colourless solid (16.4 mg, 6%)[R_(f)=0.15 (CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺, 650.1678. C₃₀H₃₁N₃O₉NaPFrequires [MNa⁺], 650.1680); ³¹P NMR (202 MHz, MeOD): ^(TM)P 2.87, 3.03;¹⁹F NMR (470 MHz, MeOD): ^(TM)F −167.95, −167.13; ¹H NMR (500 MHz,MeOD): ^(TM)H 1.37-1.42 (m, 6H, C(CH₃)₂), 1.61-1.69 (m, 1H, H-2′, onediast.), 1.79-1.87 (m, 1H, H-2′, one diast.), 2.06 (ddd, 1H, J=3.0 Hz,J=6.1 Hz, J=13.6 Hz, H-2′, one diast.), 2.15 (ddd, 1H, J=3.2 Hz, J=5.9Hz, J=13.7 Hz, H-2′, one diast.), 3.98-4.04 (m, 1H, H-4′), 4.19-4.35 (m,3H, CH₂OP, H-3′), 5.09-5.13 (m, 1H, OCHHPh), 5.18-5.19 (m, 1H, OCHHPh),6.05-6.15 (m, 1H, H-1′), 7.28-7.40 (m, 7H, ArH), 7.48-7.55 (m, 3H, ArH),7.62 (d, ³J_(H-F)=6.4 Hz, H-6, one diast.), 7.70 (d, ³J_(H-F)=6.4 Hz,H-6, one diast.), 7.86-7.90 (m, 1H, ArH), 8.17-8.22 (m, 1H, ArH); ¹³CNMR (125 MHz, MeOD): ^(TM)C 27.5 (d, ³J_(C-P)=4.4 Hz, CH₃), 27.9 (d,³J_(C-P)=7.3 Hz, CH₃), 28.0 (d, ³J_(C-P)=7.3 Hz, CH₃), 40.7 (CH₂), 40.8(CH₂), 65.2 (C), 67.8 (d, ²J_(C-P)=6.5 Hz, CH₂), 68.3 (CH₂), 72.0 (CH),72.1 (CH), 86.6 (d, ³J_(C-P)=8.2 Hz, CH), 86.8 (d, ³J_(C-P)=7.8 Hz, CH),86.9 (CH), 116.3 (d, ³J_(C-P)=3.2 Hz, CH), 116.7 (d, ³J_(C-P)=2.9 Hz,CH), 122.8 (CH), 122.9 (CH), 125.4 (CH), 125.5 (CH), 125.6 (CH), 126.0(CH), 126.1 (CH), 126.4 (CH), 126.5 (CH), 127.4 (CH), 127.5 (CH), 127.7(CH), 127.8 (CH), 127.9 (C), 128.0 (CH), 128.9 (CH), 129.3 (CH), 129.4(CH), 129.6 (CH), 136.2 (C), 137.3 (C), 141.8 (d, ¹J_(C-F)=234.4 Hz, C),147.9 (d, ³J_(C-P)=7.7 Hz, C), 148.0 (d, ³J_(C-P)=8.2 Hz, C), 150.7 (d,⁴J_(C-F)=3.7 Hz, C), 159.5 (d, ²J_(C-F)=25.8 Hz, C), 159.6 (d,²J_(C-F)=25.8 Hz, C), 176.5 (C), 176.6 (C), m/z (ES) 650.0 (MNa⁺, 100%);Reverse-phase HPLC eluting with H₂O/MeOH from 100/0 to 0/100 in 45minutes, 1 ml/min, L=275 nm, showed two peaks of the diastereoisomerswith t_(R) 20.80 min. and t_(R) 21.00 min. (72%: 24%).

5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl(ethoxy-α,α-dimethylglycine)]phosphate (CPF396)

The phosphoramidate was prepared using 5-fluoro-2′-deoxyuridine (0.40 g,1.62 mmol), tert-butylmagnesium chloride in tetrahydrofuran (^(t)BuMgCl)(1.0 M, 2.43 mL, 2.43 mmol) and 1-naphthyl(ethoxy-α,α-dimethylglycine)phosphorochloridate (1.14 g, 3.20 mmol) according to general procedureE. Purification by gradient column chromatography eluting with CH₂Cl₂until CH₂Cl₂-MeOH (95:5) afforded the title compound as a colourlesssolid (54.0 mg, 2%) [R_(f)=0.10 (CH₂Cl₂-MeOH, 95:5)], (Found: MNa⁺,588.1528. C₂₅H₂₉N₃₀NaPF requires [MNa⁺], 588.1523); ³¹P NMR (202 MHz,MeOD): ^(TM)P 2.91, 3.03; ¹⁹F NMR (470 MHz, MeOD): ^(TM)F −167.38,−167.21; ¹H NMR (500 MHz, MeOD): ^(TM)H 1.24 (t, 3H, J=7.1 Hz, CH₂CH₃,one diast.), 1.25 (t, 3H, J=7.1 Hz, CH₂CH₃, one diast.), 1.50-1.55 (m,6H, C(CH₃)₂), 1.68-1.76 (m, 1H, H-2′, one diast.), 1.87-1.94 (m, 1H,H-2′, one diast.), 2.09 (ddd, 1H, J=2.9 Hz, J=6.3 Hz, J=13.4 Hz, H-2′,one diast.), 2.19 (ddd, 1H, j=3.0 Hz, =6.3 Hz, j=13.8 Hz, H-2′, onediast.), 4.07-4.10 (m, 1H, H-4′), 4.16 (q, 2H, j=7.1 Hz, CH₂CH₃),4.36-4.41 (m, 3H, CH₂OP, H-3′), 6.10-6.18 (m, 1H, H-1′), 7.40-7.46 (m,1H, ArH), 7.50-7.59 (m, 3H, ArH), 7.66-7.72 (m, 2H, ArH, H-6), 7.85-7.91(m, 1H, ArH), 8.18-8.24 (m, 1H, ArH); ¹³C NMR (125 MHz, MeOD): ^(TM)C14.4 (CH₃), 27.5 (br s, CH₃), 27.9 (d, ³J_(C-P)=6.1 Hz, CH₃), 28.0 (d,³J_(C-P)=6.1 Hz, CH₃), 40.7 (CH₂), 40.8 (CH₂), 58.2 (C), 58.3 (C), 62.6(CH₂), 67.8 (d, ²J_(C-P)=4.9 Hz, CH₂), 67.9 (d, ²J_(C-P)=4.5 Hz, CH₂),72.0 (CH), 72.1 (CH), 86.7 (d, ³J_(C-P)=7.7 Hz, CH), 86.9 (d,³J_(C-P)=7.3 Hz, CH), 87.0 (CH), 116.3 (d, ³J_(C-P)=3.2 Hz, CH), 116.6(d, ³J_(C-P)=2.9 Hz, CH), 122.8 (CH), 122.9 (CH), 125.4 (CH), 125.6(CH), 125.7 (CH), 126.0 (CH), 126.1 (CH), 126.5 (CH), 127.4 (CH), 127.5(CH), 127.7 (CH), 127.8 (CH), 127.9 (C), 128.0 (C), 128.9 (CH), 136.2(C), 141.8 (d, ¹J_(C-F)=233.5 Hz, C), 148.0 (d, ²J_(C-P)=7.3 Hz, C),148.1 (d, ²J_(C-P)=7.6 Hz, C), 150.5 (C), 150.6 (C), 159.3 (d,²J_(C-F)=26.2 Hz, C), 159.4 (d, ²J_(C-F)=26.6 Hz, C), 176.8 (C), 176.9(C); m/z (ES) 588.1 (MNa⁺, 100%); Reverse-phase HPLC eluting withH₂O/MeOH from 100/0 to 0/100 in 45 minutes, 1 ml/min, L=275 nm, showedone peak of the mixture of diastereoisomers with t_(R) 16.05 min. (96%).

5-Fluoro-2′-deoxyuridine-5′-O-[phenyl(benzoxy-L-prolinyl)] phosphate(CPF583)

Prepared according to the standard procedure D from5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol,0.40 mL) and phenyl(benzoxy-L-prolinyl)-phosphochloridate (0.77 g, 2.03mmol) in THF (10 mL). Column purification followed by two preparativeTLC purifications gave the product as a white solid (0.010 g, 2%).

³¹P-NMR (MeOD, 202 MHz) δ 1.82

¹⁹F-NMR (MeOD, 470 MHz) δ −167.91

¹H-NMR (MeOD, 500 MHz) δ 7.84 (d, j=7.18 Hz, 1H, H-base), 7.39-7.33 (m,7H, H—Ar), 7.22-7.19 (m, 3H, H—Ar), 6.26-6.23 (m, 1H, H-1′), 5.22-5.13(m, CH₂Ph ester), 4.40-4.35 (m, 3H, NCH, 2×H-5′), 4.33-4.28 (m, 1H,H-3′), 4.06-4.04 (m, 1H, H-4′), 3.36-3.32 (m, 2H, NCH₂), 2.26-2.19 (m,1H, H-2′), 2.18-2.13 (m, 1H, CH₂-L-Pro), 2.00-1.81 (m, 4H, 3×H,CH₂-L-Pro, 1×H, H-2′)

¹³C-NMR (MeOD, 125 MHz) δ 174.81 (C═O, ester), 159.40 (C═O, base), 152.0(d, ²J_(C-P)=6.32 Hz, OC—Ar), 150.71 (C═O, base), 141.88 (¹J_(C-F)=232Hz, CF, base), 137.23 (C—Ar), 131.33, 129.70, 129.48, 129.45, 129.30,126.45 (CH—Ar), 125.80, 125.53 (2×d, ²J_(C-F)=29.0 Hz, CH-base), 121.00,120.96 (CH—Ar), 87.80 (C-1′), 86.80 (C-4′), 72.02 (C-3′), 68.16 (CH₂Ph),67.64 (d, ²J_(C-P)=4.65 Hz, C-5′), 62.40 (d, ²J_(C-P)=5.60 Hz, NCH),48.03 (d, ²J_(C-P)=4.80 Hz, NCH₂), 41.07 (C-2′), 32.18, 32.11(CH₂-L-Pro), 26.29, 26.21 (CH₂-L-Pro).

MS (ES+) m/e: 612 (MNa⁺, 100%), 590 (MH⁺, 1%) Accurate mass:C₂₇H₂₉FN₃O₉P required 589.51

5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl(benzoxy-L-prolinyl)] phosphate(CPF577)

Prepared according to the standard procedure D from5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol,0.40 mL) and 1-naphthyl(benzoxy-L-prolinyl)-phosphochloridate (0.84 g,2.03 mmol) in THF (10 mL). Column purification followed by twopreparative TLC purifications gave the product as a white solid (0.006g, 1%).

³¹P-NMR (MeOD, 202 MHz) δ 2.27

¹⁹F-NMR (MeOD, 121 MHz) δ −167.46

¹H-NMR (MeOD, 500 MHz) δ 8.14-8.12 (m, 1H, H—Ar), 7.90-7.89 (m, 1H,H—Ar), 7.74-7.71 (m, 2H, 1×H—Ar, 1×H-base), 7.56-7.42 (m, 4H, H—Ar),7.36-7.33 (m, 5H, H—Ar), 6.13 (t, =6.38 Hz, H-1′), 5.22-5.13 (m, 2H,CH₂Ph), 4.49-4.46 (m, 1H, NCH), 4.42-4.33 (m, 2H, H-5′), 4.25-4.23 (m,1H, H-3′), 4.06-4.04 (m, 1H, H-4′), 3.36-3.34 (m, 2H, NCH₂), 2.23-2.15(m, 1H, CH₂-L-Pro), 2.10-2.02 (m, 2H, 1×H, CH₂-L-Pro, 1×H, H-2′),1.97-1.77 (m, 2H, CH₂-L-Pro), 1.63-1.57 (m, 1H, H-2′)

¹³C-NMR (MeOD, 125 MHz) δ 174.82 (C═O, ester), 159.52 (C═O, base),150.54 (C═O, base), 147.84, 147.78 (d, ²J_(C-P)=6.03 Hz, OC—Ar), 141.75,139.97 (2×d, ¹J_(C-F)=232 Hz, CF, base), 137.20, 136.34 (C—Ar), 129.76,129.65, 129.44, 129.36, 129.27, 129.06, 128.95, 128.04, 128.75, 126.56(CH—Ar), 125.41 (d, ²J_(C-F)=30.0 Hz, CH-base), 122.13 (CH—Ar), 115.76(d, ³J_(C-P)=3.3 Hz, CH—Ar), 87.06 (C-1′), 86.79 (C-4′), 72.23 (C-3′),68.15 (d, ²J_(C-P)=5.46 Hz, C-5′), 68.08 (CH₂Ph), 62.53 (d,²J_(C-P)=5.60 Hz, NCH), 48.26 (d, ²J_(C-P)=5.34 Hz, NCH₂), 40.97 (C-2′),32.16, 32.09 (CH₂-L-Pro), 26.22, 26.15 (CH₂-L-Pro).

5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl(3,3-dimethyl-1-butoxy-L-alaninyl)]phosphate (CPF585)

Prepared according to the standard procedure D from5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol,0.40 mL) and1-naphthyl-(3,3-dimethyl-1-butoxy-L-alaninyl)-phosphochloridate (1.21 g,3.04 mmol) in THF (10 mL). Column purification followed by twopreparative TLC purifications gave the product as a white solid (0.010g, 2%).

³¹P-NMR (MeOD, 202 MHz) δ 4.48, 4.33

¹⁹F-NMR (MeOD, 470 MHz) δ −167.30, −167.47

¹H-NMR (MeOD, 500 MHz) δ 8.20-8.17 (m, 1H, H—Ar), 7.91-7.89 (m, 1H,H—Ar), 7.77-7.72 (m, 2H, H—Ar), 7.58-7.51 (m, 3H, H-base, 2×H—Ar),7.46-7.41 (2×t, 1H, =7.8 Hz, H—Ar), 6.19-6.13 (m, 1H, H-1′), 4.42-4.40(m, 1H, 1×H-5′), 4.38-4.32 (m, 2H, H-3′, 1×H-5′), 4.14-4.00 (m, 4H,H-4′, CHCH₃, OCH₂CH₂(CH₃)₃), 2.21-2.13 (m, 1H, 1×H-2′), 1.91-1.76 (m,1H, 1×H-2′), 1.52-1.48 (m, 2H, OCH₂CH₂(CH₃)₃), 1.37-1.35 (m, 3H, CHCH₃),0.92, 0.91 (2×s, 9H, OCH₂CH₂(CH₃)₃)

¹³C-NMR (MeOD, 125 MHz) δ 175.16, 174.84 (2×d, ³J_(C-P)=4.75 Hz, C═O,ester), 159.56, 159.35 (C═O, ester), 150.61 (C═O, ester), 148.00, 147.86(2×d, ²J_(C-P)=6.25 Hz, OC—Ar), 141.78, 141.73 (2×d, ¹J_(C-F)=232 Hz,CF, base), 136.28 (C—Ar), 128.98, 128.95, 127.92, 127.90, 127.58,126.57, 126.20, 126.14 (CH—Ar), 125.63, 125.55 (2×d, ²J_(C-F)=34 Hz, CH,base), 122.65, 122.63 (CH—Ar), 116.48, 116.15 (2×d, ³J_(C-P)=3.0 Hz,CH—Ar), 87.01, 86.94 (C-1′), 86.73, 86.68 (d, ³J_(C-P)=7.75 Hz, C-4′),72.18, 72.07 (C-3′), 67.87, 67.85 (2×d, ²J_(C-P)=5.0 Hz, C-5′), 64.08,64.05 (OCH₂CH₂(CH₃)₃), 51.86 (d, ³J_(C-P)=5.5 Hz, CHCH₃), 42.74(OCCH₂CH₂(CH₃)₃), 40.91, 40.83 (C-2′), 29.96 (OCH₂CH₂(CH₃)₃), 20.50,20.34 (2×d, ³J_(C-P)=6.5 Hz, CHCH₃).

MS (ES+) m/e: 630 (MNa⁺, 100%), 608 (MH⁺, 10%) Accurate mass:C₂₈H₃₅FN₃O₉P required 607.56

5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(cyclobutoxy-L-alaninyl)]phosphate(CPF578)

Prepared according to the standard procedure D from5-Fluoro-2′-deoxyuridine (0.23 g, 0.93 mmol), NMI (0.38 g, 4.67 mmol,0.37 mL) and 1-naphthyl-(cyclobutoxy-L-alaninyl)-phosphochloridate (0.85g, 2.33 mmol) in THF (10 mL). Column purification followed bypreparative TLC purification gave the product as a white solid (0.010 g,2%). ³¹P-NMR (MeOD, 202 MHz) δ 4.54, 4.36

¹⁹F-NMR (MeOD, 470 MHz) δ−167.12, −167.29

¹H-NMR (MeOD, 500 MHz) δ 8.18-8.17 (m, 1H, H—Ar), 7.81-7.87 (m, 1H,H—Ar), 7.74-7.71 (m, 2H, 1×H—Ar, 1×H-base), 7.60-7.53 (m, 3H, H—Ar),7.46-7.43 (2×t, J=8.0 Hz, 1H, H—Ar), 6.18-6.12 (m, 1H, H-1′), 5.00-4.95(m, 1H, OCH ester), 4.41-4.36 (m, 3H, 2×H-5′, H-3′), 4.11-4.00 (m, 2H,H-4′, CHCH₃), 2.36-2.27 (m, 2H, CH₂), 2.18-1.98 (m, 3H, CH₂ ester,1×H-2′), 1.82-1.56 (m, 3H, CH₂ ester, 1×H-2′), 1.36-1.34 (m, 3H, CHCH₃)

¹³C-NMR (MeOD, 125 MHz) δ 175.97, 173.34 (C═O, ester), 159.88 (C═O,base), 151.64 (C═O, base), 146.58 (OC—Ar), 141.15 (d, ¹J_(C-F)=220 Hz,CF, base), 136.28 (C—Ar), 128.93, 127.89, 127.54, 126.52, 126.18, 126.14(CH—Ar), 125.53, 125.44 (2×d, ²J_(C-F)=32.5 Hz, CH-base), 122.63(CH—Ar), 116.46, 116.44 (2×d, ³J_(C-P)=2.5 Hz, CH—Ar), 86.98 (d,³J_(C-P)=6.25 Hz, C-4′), 86.71 (C-1′), 72.14, 72.04 (C-3′), 71.07 (OCHester), 67.83 (d, 2J_(C-P)=7.38 Hz, C-5′), 51.66 (d, ²J_(C-P)=8.75 Hz,CHCH₃), 40.89, 40.83 (C-2′), 31.03 (OCHCH₂), 20.43 (CHCH₃), 14.23 (CH₂ester).

MS (ES+) m/e: 600 (MNa⁺, 100%), 578 (MH⁺, 10%) Accurate mass:C₂₆H₂₉FN₃O₉P required 577.50

5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(cyclopropylmethanoxy-L-alaninyl)]phosphate (CPF579)

Prepared according to the standard procedure D from5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol,0.40 mL) and1-naphthyl-(cyclopropylmethanoxy-L-alaninyl)-phosphochloridate (0.93 g,2.54 mmol) in THF (10 mL). Column purification gave the product as awhite solid (0.056 g, 10%).

³¹P-NMR (MeOD, 202 MHz) δ 4.58, 4.30

¹⁹F-NMR (MeOD, 470 MHz) δ −167.18, −167.22

¹H-NMR (MeOD, 500 MHz) δ 8.18 (d, J=7.0 Hz, 1H, H—Ar), 7.89-7.87 (m, 1H,H—Ar), 7.73-7.70 (m, 2H, H—Ar), 7.58-7.53 (m, 3H, H—Ar), 7.45-7.40 (2×t,J=8.0 Hz, 1H, H—Ar), 6.17-6.11 (m, 1H, H-1′), 4.43-4.41 (m, 1H, H-5′),4.38-4.32 (m, 2H, H-5′, H-3′), 4.11-4.04 (m, 2H, H-4′, CHCH₃), 3.95-3.85(m, 2H, OCH₂ ester), 2.19-2.11 (m, 1H, H-2′), 1.84-1.72 (m, 1H, H-2′),1.38, 1.36 (2×d, J=5.0 Hz, 3H, CHCH₃), 1.15-1.07 (m, 1H, OCH₂CH ester),0.59-0.50 (m, 2H, CH₂ ester), 0.30-0.24 (m, 2H, CH₂ ester)

¹³C-NMR (MeOD, 125 MHz) δ 175.25, 174.94 (2×d, ³J_(C-P)=4.75 Hz, C═O,ester), 159.54, 159.35 (C═O, base), 150.60, 150.56 (C═O, base), 148.05,147.86 (2×d, ²J_(C-P)=7.5 Hz, OC—Ar), 141.79, 141.73 (2×d, ¹J_(C-F)=232Hz, CF, base), 136.29 (C—Ar), 128.94 (d, ³J_(C-P)=4.4 Hz, CH—Ar), 127.89(d, ⁴J_(C-P)=3.7 Hz, CH—Ar), 127.56, 126.55, 126.52, 126.19, 126.16(CH—Ar), 125.64, 125.53 (²J_(C-F)=34 Hz, CH-base), 122.65 (CH—Ar),116.54, 116.24 (2×d, ⁴J_(C-P)=2.6 Hz, CH—Ar), 87.04, 86.99 (C-1′),86.90, 86.73 (2×d, ³J_(C-P)=7.1 Hz, C-4′), 72.18, 72.07 (C-3′), 71.21,71.18 (OCH₂, ester), 67.87, 67.84 (apparent t, ²J_(C-P)=5.0 Hz, C-5′),51.88 (d, ²J_(C-P)=10.0 Hz, CHCH₃), 40.91, 40.83 (C-2′), 20.60, 20.46(2×d, ³J_(C-P)=6.5 Hz, CHCH₃), 10.69 (OCH₂CH ester), 3.70, 3.65 (2×CH₂,ester).

MS (ES+) m/e: 600 (MNa⁺, 100%), 578 (MH⁺, 15%) Accurate mass:C₂₆H₂₉FN₃O₉P required 577.50.

HPLC_(b) (H₂O/Acetonitrile from 100/0 to 0/100 in 35 min) Rt 12.91 min.

5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(tetrahydropyroxy-L-alaninyl)]phosphate(CPF580)

Prepared according to the standard procedure E from5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), tBuMgCl (1.1 mL, 1.1 mmol)and 1-naphthyl-(tetrahydropyroxy-L-alaninyl)-phosphochloridate (0.80 g,2.03 mmol) in THF (10 mL). Column purification followed by twopreparative TLC purifications gave the product as a white solid (0.010g, 1.6%).

³¹P-NMR (MeOD, 202 MHz) δ 3.77, 3.22

¹⁹F-NMR (MeOD, 470 MHz) δ −168.27, −168.35

¹H-NMR (MeOD, 500 MHz) δ 8.60 (d, J=7.0 Hz, 2H, H—Ar), 8.22-8.19 (m, 1H,H—Ar), 7.92-7.91 (d, =5.50 Hz, 1H, H—Ar), 7.60-7.45 (m, 4H, H—Ar,H-base), 6.29-6.25 (m, 1H, H-1′), 5.25-5.17 (m, 1H, H-3′), 4.96-4.87 (m,1H, CH-ester), 4.28-4.26 (m, 1H, H-4′), 4.11-4.03 (m, 1H, CHCH₃),3.88-3.66 (m, 4H, 2×OCH_(2a′) ester, 2×H-5′), 3.55-3.50 (m, 2H,2×OCH_(2a″) ester), 2.63-2.30 (m, 2H, H-2′), 1.91-1.85 (m, 2H,2×CH_(2b′) ester), 1.65-1.54 (m, 2H, CH_(2b″) ester), 1.39-1.35 (m, 3H,CHCH₃).

¹³C-NMR (MeOD, 125 MHz) δ 174.34 (C═O, ester), 159.24 (C═O, base),150.76 (C═O, base), 148.03 (OC—Ar), 141.97 (d, ¹J_(C-F)=238 Hz, CF,base), 136.37 (C—Ar), 128.97, 128.56, 127.61, 127.57, 126.58, 126.23,126.16, 126.12, 125.84 (CH—Ar), 122.70 (d, ²J_(C-F)=24.0 Hz, CH-base),116.62, 116.37 (CH—Ar), 87.54 (d, ³J_(C-P)=5.40 Hz, C-4′), 86.60, 86.57(C-1′), 79.82, 79.47 (C-3′), 71.45 (CH-ester), 66.12, 66.08 (2×OCH₂aester), 66.02 (C-5′), 51.83 (CHCH₃), 39.97, 39.94 (C-2′), 32.65, 32.57(2×CH₂b ester), 20.45, 20.30 (CHCH₃).

MS (ES+) m/e: 630 (MNa⁺, 100%), 608 (MH⁺, 10%) Accurate mass:C₂₇H₃₁FN₃O₁₀P required 607.52.

5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(pentoxy-L-alaninyl)]phosphate (CPF581)

Prepared according to the standard procedure E from5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), tBuMgCl (1.1 mL, 1.1 mmol)and 1-naphthyl-(pentoxy-L-alaninyl)-phosphochloridate (0.78 g, 2.03mmol) in THF (10 mL). Column purification gave the product as a whitesolid (0.047 g, 8%).

³¹P-NMR (MeOD, 202 MHz) δ 4.48, 4.32

¹⁹F-NMR (MeOD, 470 MHz) δ −167.18, −167.29

¹H-NMR (MeOD, 500 MHz) δ 8.25-8.17 (m, 1H, H—Ar), 8.05-7.95 (m, 2H,H—Ar), 7.85-7.60 (m, 2H, H—Ar, H-base), 7.65-7.48 (m, 3H, H—Ar),6.30-6.18 (m, 1H, H-1′), 4.60-4.37 (m, 3H, 2×H-5′, H-3′), 4.28-4.00 (m,4H, H-4′, CHCH₃, OCH₂CH₂CH₂CH₂CH₃), 2.32-2.12 (m, 1H, H-2′), 1.95-1.75(m, 1H, H-2′), 1.70-1.55 (m, 2H, OCH₂CH₂CH₂CH₂CH₃), 1.50-1.28 (m, 7H,4×H OCH₂CH₂CH₂CH₂CH₃, CHCH₃), 0.83, 0.82 (2×d, J=7.9 Hz, 3H,OCH₂CH₂CH₂CH₂CH₃)

¹³C-NMR (MeOD, 125 MHz) δ 175.22, 174.91 (C═O, ester), 159.5 (C═O,base), 150.54 (C═O, base), 147.90, 147.88 (OC—Ar), 141.75 (d,¹J_(C-F)=225 Hz, CF, base), 136.37 (C—Ar), 128.95, 127.90, 127.56,126.55, 126.19 (CH—Ar), 125.64, 125.53 (2×d, ²J_(C-F)=34.0 Hz, CH-base),122.65 (CH—Ar), 116.51, 116.21 (CH—Ar), 87.03, 86.96 (C-1′), 86.85,86.74 (C-4′), 72.16, 72.05 (C-3′), 67.87 (d, ²J_(C-P)=5.0 Hz, C-5′),66.54 (OCH₂), 51.87, 51.81 (d, ²J_(C-P)=7.5 Hz, CHCH₃), 40.87, 40.80(C-2′), 29.35, 29.10 (CH₂ ester), 23.33 (CH₂ ester), 20.60, 20.43 (2×d,³J_(C-P)=6.5 Hz, CHCH₃), 14.28 (CH₃, ester). MS (ES+) m/e: 616 (MNa⁺,100%), 594 (MH⁺, 10%) Accurate mass: C₂₇H₃₃FN₃O₉P required 593.54.

HPLC_(b) (H₂O/Acetonitrile from 100/0 to 0/100 in 35 min) Rt 15.56 min.

5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(cyclopentoxy-L-alaninyl)]phosphate(CPF582)

Prepared according to the standard procedure E from5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), tBuMgCl (1.1 mL, 1.1 mmol)and 1-naphthyl-(cyclopentoxy-L-alaninyl)-phosphochloridate (0.77 g, 2.03mmol) in THF (10 mL). Column purification gave the product as a whitesolid (0.030 g, 5%).

³¹P-NMR (MeOD, 202 MHz) δ 4.53, 4.37

¹⁹F-NMR (MeOD, 470 MHz) δ −167.07, −167.19

¹H-NMR (MeOD, 500 MHz) δ 8.18-8.16 (m, 1H, H—Ar), 7.89-7.85 (m, 1H,H—Ar), 7.70 (apparent t, J=6.50 Hz, 2H, H—Ar), 7.57-7.50 (m, 3H, 2×H—Ar,H-base), 7.45-7.40 (m, 1H, H—Ar), 6.16-6.11 (m, 1H, H-1′), 5.15-5.09 (m,1H, OCH ester), 4.41-4.30 (m, 3H, 2×H-5′, H-3′), 4.11-4.08 (m, 1H,H-4′), 4.04-3.98 (m, 1H, CHCH₃), 2.19-2.10 (m, 1H, H-2′), 1.86-1.73 (m,3H, OCHCH₂ ester), 1.73-1.56 (m, 6H, H-2′, CH₂ ester), 1.35, 1.34 (2×d,J=6.57 Hz, CHCH₃)

¹³C-NMR (MeOD, 125 MHz) δ 174.68, 174.64 (C═O, ester), 159.27 (C═O,base), 150.51 (C═O, base), 147.86 (d, ²J_(C-P)=7.5 Hz, OC—Ar), 141.78,141.72 (2×d, ¹J_(C-F)=232 Hz, CF-base), 136.30 (C—Ar), 128.95, 128.54,127.94, 127.80, 127.60, 127.56, 127.17, 126.80, 126.54, 126.19, 126.16(CH—Ar), 125.66, 125.53 (2×d, ²J_(C-F)=34 Hz, CH-base), 122.65, 122.61(CH—Ar), 116.53, 116.22 (2×d, ⁴J_(C-P)=3.75 Hz, CH—Ar), 86.99, 86.96(C-1′), 86.70 (d, ³J_(C-P)=7.50 Hz, C-4′), 79.64, 79.61 (OCH ester),72.21, 72.07 (C-3′), 67.89, 67.85 (2×d, ²J_(C-P)=5.0 Hz, C-5′), 51.92(d, ²J_(C-P)=5.0 Hz, CHCH₃), 40.92, 40.86 (C-2′), 33.65, 33.61, 33.52,33.47 (2×CH₂ ester), 24.68, 24.66 (CH₂ ester), 20.45, 20.30 (2×d,³J_(C-P)=6.25 Hz, CHCH₃).

MS (ES+) m/e: 614 (MNa⁺, 100%), 592 (MH⁺, 30%) Accurate mass:C₂₇H₃₁FN₃O₉P required 591.52

HPLC_(b) (H₂O/Acetonitrile from 100/0 to 0/100 in 35 min) Rt 14.03 min.

5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(2-indanoxy-L-alaninyl)]phosphate(CPF597)

Prepared according to the standard procedure E from5-Fluoro-2′-deoxyuridine (0.30 g, 1.22 mmol), tBuMgCl (1.34 mL, 1.34mmol) and 1-naphthyl-(2-indanoxy-L-alaninyl)-phosphochloridate (1.06 g,2.43 mmol) in THF (20 mL). Column purification gave the product as awhite solid (0.045 g, 6%).

³¹P-NMR (MeOD, 202 MHz) δ 4.62, 4.30

¹⁹F-NMR (MeOD, 470 MHz) δ −167.14, −167.34

¹H-NMR (MeOD, 500 MHz) δ 8.15-8.12 (m, 1H, H—Ar, Naph), 7.89-7.87 (m,1H, H—Ar, Naph), 7.72-7.67 (m, 2H, H—Ar, Naph), 7.56-7.46 (m, 3H,2×H—Ar, H-base), 7.40-7.37 (m, 1H, H—Ar), 7.20-7.12 (m, 4H, H—Ar, Ph),6.14-6.08 (m, 1H, H-1′), 5.49-5.46 (m, 1H, OCH ester), 4.32-4.26 (m, 3H,2×H-5′, H-3′), 4.04-3.98 (m, 1H, H-4′, CHCH₃), 3.30-3.24 (m, 2H, 2×CHester), 2.99-2.91 (m, 2H, 2×CH ester), 2.14-2.07 (m, 1H, H-2′),1.75-1.64 (m, 1H, H-2′), 1.33-1.29 (m, 3H, CHCH₃)

¹³C-NMR (MeOD, 125 MHz) δ 175.02, 174.66 (2×d, ³J_(C-P)=3.75 Hz, C═O,ester), 159.48 (²J_(C-F)=25.0 Hz, C═O, base), 150.57 (C═O, base),147.97, 147.80 (2×d, ²J_(C-P)=7.5 Hz, OC—Ar), 141.73, 141.68 (2×d,¹J_(C-F)=232.5 Hz, CF-base), 141.54, 141.49, 141.48, 139.10, 136.27,136.26 (C—Ar), 129.01, 128.94, 128.91, 127.91, 127.87, 128.85, 127.80,127.77, 127.60, 127.57, 127.50, 126.20, 126.18, 125.69 (CH—Ar), 125.50,125.43 (2×d, ²J_(C-F)=25 Hz, CH-base), 122.64, 122.60, 121.85 (CH—Ar),116.57, 116.26 (2×d, ⁴J_(C-P)=2.5 Hz, CH—Ar), 86.96 (C-1′), 86.87, 86.66(2×d, ³J_(C-P)=7.50 Hz, C-4′), 77.85, 79. (OCH ester), 72.21, 72.07(C-3′), 67.77, 67.75 (2×d, ²J_(C-P)=6.25 Hz, C-5′), 51.97, 51.82(CHCH₃), 40.91, 40.86 (C-2′), 40.44, 40.43, 40.38, 40.34 (2×CH₂ ester),20.30, 20.16 (2×d, ³J_(C-P)=6.25 Hz, CHCH₃)

5-Fluoro-2′-deoxyuridine-5′-O-[phenyl-(benzoxy-L-methioninyl)] phosphate(CPF586)

Prepared according to the standard procedure D from5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol,0.40 mL) and phenyl-(benzoxy-L-methioninyl)-phosphochloridate (0.7 g,mmol) in THF (10 mL). Column purification gave the product as ayellowish solid (0.014 g, 2%).

³¹P-NMR (MeOD, 202 MHz) δ 4.34, 3.94

¹⁹F-NMR (MeOD, 470 MHz) δ −167.40, −167.69

¹H-NMR (MeOD, 500 MHz) δ 7.83-7.80 (m, 1H, H—Ar), 7.74-7.72 (m, 1H,H—Ar), 7.64-7.62 (m, 1H, H—Ar), 7.37-7.32 (m, 6H, H—Ar, H-base),7.26-7.17 (m, 2H, H—Ar), 6.25-6.17 (m, 1H, H-1′), 5.18, 5.13 (AB system,J_(AB)=12.0 Hz, 2H, CH₂Ph), 4.40-4.35 (m, 1H, H-3′), 4.32-4.22 (m, 2H,H-5′), 4.16-4.03 (m, 2H, NHCH, H-4′), 2.44, 2.36 (2×t, J=7.50 Hz, CH₂S),2.16-2.08 (m, 1H, 1×H-2′), 1.98-1.82 (m, 6H, 1×H-2′, NHCHCH₂CH₂SCH₃),

MS (ES+) m/e: 646 (MNa⁺, 100%), 624 (MH⁺, 10%) Accurate mass:C₂₇H₃₁FN₃O₉PS required 623.56

5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(benzoxy-L-phenylalaninyl)]phosphate(CPF587)

Prepared according to the standard procedure D from5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol,0.40 mL) and 1-naphthyl-(benzoxy-L-phenylalaninyl)-phosphochloridate(1.45 g, mmol) in THF (10 mL). Column purification gave the product as awhite solid (0.007 g, 1%).

³¹P-NMR (MeOD, 202 MHz) δ 4.27, 4.14

¹⁹F-NMR (MeOD, 470 MHz) δ −166.99, −167.18

¹H-NMR (MeOD, 500 MHz) δ 8.11-8.00 (m, 1H, H—Ar, Ar), 7.89-7.85 (m, 1H,H—Ar), 7.69-7.67 (m, 1H, H—Ar), 7.60-7.49 (m, 3H, 2×H—Ar, H-base),7.37-7.33 (m, 2H, H—Ar), 7.25-7.12 (m, 10H, H—Ar), 6.09-6.04 (m, 1H,H-1′), 5.11-5.01 (m, 2H, CH₂Ph), 4.29-4.18 (m, 1H, CHCH₃), 4.15-4.08 (m,1H, H-3′), 4.02-3.95 (m, 2H, H-5′), 3.86-3.67 (m, 1H, H-4′), 3.14-3.10(m, 1H, 1×NHCHCH₂Ph), 2.91-2.82 (m, 1H, 1×NHCHCH₂Ph), 2.12-2.06,2.00-1.95 (2×m, 1H, H-2′), 1.68-1.62, 1.42-1.36 (2×m, 1H, H-2′)

5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(2,2-dimethylpropoxy-L-alaninyl)]phosphate (CPF588)

Prepared according to the standard procedure D from5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol,0.40 mL) and1-naphthyl-(2,2-dimethylpropoxy-L-alaninyl)-phosphochloridate (0.77 g,mmol) in THF (10 mL). Column purification gave the product as a whitesolid (0.006 g, 1%).

³¹P-NMR (MeOD, 202 MHz) δ 4.56, 4.33

¹⁹F-NMR (MeOD, 470 MHz) δ −167.32, −167.43

¹H-NMR (MeOD, 500 MHz) δ 8.19-8.16 (m, 1H, H—Ar, Ar), 7.91-7.89 (m, 1H,H—Ar), 7.74-7.71 (m, 2H, H—Ar), 7.57-7.51 (m, 3H, 2×H—Ar, H-base),7.46-7.41 (m, 1H, H—Ar), 6.17-6.10 (m, 1H, H-1′), 4.42-4.30 (m, 3H,H-3′, 2×H-5′), 4.13-4.07 (m, 2H, H-4′, CHCH₃), 3.86, 3.75 (AB system,J_(AB)=10.50 Hz, 2H, CH₂C(CH₃)₃), 2.18-2.10 (m, 1H, H-2′), 1.81-1.70 (m,1H, H-2′), 1.41-1.38 (m, 3H, CHCH₃), 0.95, 0.94 (2×s, 9H, CH₂C(CH₃)₃)

5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(butoxy-L-alaninyl)] phosphate(CPF589)

Prepared according to the standard procedure D from5-Fluoro-2′-deoxyuridine (0.25 g, 1.01 mmol), NMI (0.41 g, 5.07 mmol,0.40 mL) and 1-naphthyl-(butoxy-L-alaninyl)-phosphochloridate (0.75 g,mmol) in THF (10 mL). Column purification gave the product as a whitesolid (0.006 g, 1%).

³¹P-NMR (MeOD, 202 MHz) δ 4.52, 4.35

¹⁹F-NMR (MeOD, 470 MHz) δ− 167.36, −167.49

¹H-NMR (MeOD, 500 MHz) δ 8.19-8.16 (m, 1H, H—Ar, Naph), 7.1-7.89 (m, 1H,H—Ar, Naph), 7.75-7.72 (m, 2H, H—Ar, Naph), 7.58-7.51 (m, 3H, 2×H—Ar,H-base), 7.46-7.41 (m, 1H, H—Ar), 6.18-6.11 (m, 1H, H-1′), 4.42-4.40 (m,1H, 1×H-5′), 4.37-4.32 (m, 2H, 1×H-5′, H-3′), 4.12-4.01 (m, 4H, H-4′,CHCH₃, OCH₂ CH₂CH₂CH₃), 2.20-2.12 (m, 1H, H-2′), 1.85-1.73 (m, 1H,H-2′), 1.61-1.54 (m, 2H, OCH₂CH₂CH₂CH₃), 1.39-1.31 (m, 5H,OCH₂CH₂CH₂CH₃, CHCH₃), 0.93-0.89 (m, 3H, OCH₂CH₂CH₂CH₃)

Biological Assays

Experimental data having regard to compounds embodying the presentinvention are described below.

Cell Cultures

Murine leukaemia L1210/0 and human T-lymphocyte CEM/0 cells wereobtained from the American Type Culture Collection (ATCC) (Rockville,Md.). Human glioblastoma U87 cells were kindly provided by Dr. E. Menue(Institut Pasteur, Paris, France). Thymidine kinase-deficient CEM/TK⁻cells were a kind gift from Prof. S. Eriksson (currently at UppsalaUniversity, Uppsala, Sweden) and Prof A. Karlsson (Karolinska Institute,Stockholm, Sweden). Thymidine kinase-deficient L1210/TK⁻ were derivedfrom L1210/0 cells after selection for resistance against5-bromo-2′-dUrd (Balzarini et al., 1982). Infection of relevant celllines with Mycoplasma hyorhinis (ATCC) resulted in chronically-infectedcell lines further referred to as L1210.Hyor and U87.Hyor. All cellswere maintained in Dulbecco's modified Eagle's medium (DMEM)(Invitrogen, Carlsbad, Calif.) with 10% foetal bovine serum (FBS)(Biochrom AG, Berlin, Germany), 10 mM Hepes and 1 mM Sodium Pyruvate(Invitrogen). Cells were grown at 37° C. in a humidified incubator witha gas phase of 5% CO₂.

Cytostatic Assays

Monolayer cells (U87 and U87.Hyor) were seeded in 48-well microtiterplates (Nunc™, Roskilde, Denmark) at 10,000 cells/well. After 24 hours,an equal volume of fresh medium containing the test compounds was added.On day 5, cells were trypsinized and counted in a Coulter counter(Analis, Suarlée, Belgium). Suspension cells (L1210/0, L1210/TK⁻,L1210.Hyor, CEM/0, CEM/TK⁻) were seeded in 96-well microtiter plates(Nunc™) at 60,000 cells/well in the presence of a given amount of thetest compounds. The cells were allowed to proliferate for 48 h (L1210)or 72 hours (CEM) and were then counted in a Coulter counter. The 50%inhibitory concentration (IC₅₀) was defined as the compoundconcentration required to reduce the number of viable cells by 50%.

Assay 1.

The samples were assayed for biological activity versus a range oftumour cell lines with data recorded in Table 1 below. Data areexpressed as CC₅₀ in M, i.e. cytostatic concentration required toinhibit cell proliferation by 50%. The cell lines employed were L1210/0(a leukemia cell line), FM3A/0 (a breast cancer cell line), Cem/0 (anacute lymphoblastic leukemia cell line) and HeLa (a cervical cell line).

Table 1 also contains comparative data for 5FU, 5-FdUrd and referencecompounds CPF 382, CPF 437 and CPF 438. The structure of CPF 382 isgiven above. The structure of each of CPF 437 and CPF 438 is as follows:

As can be seen from the data in Table 1, compounds of the presentinvention can exhibit cytostatic activity that is comparable to orbetter than that of 5-FU, whilst exhibiting marked cytostatic activityin nucleoside kinase-deficient cells. Such a cytostatic activity innucleotide kinase-deficient cells is in direct contrast to that of5-FdUrd.

As can also be seen from Table 1, the activity in TK⁻ cells of compoundsembodying the present invention can be markedly greater than that ofreference compounds CPF 382, CPF 437 and CPF 438.

TABLE 1 L1210/0 L1210/TK⁻ FM3A/0 FM3A/TK⁻ Cem/0 Cem/TK⁻ HeLa HeLa/TK⁻5-FdUrd 0.00082 ± 0.00008 3.1 ± 0.2 0.028 ± 0.002 1.5 ± 0.1 5-FdUrd (2)0.0010 ± 0.0001 4.8 ± 4.0 0.0065 ± 0.0055 0.70 ± 0.02 0.026 ± 0.000 4.4± 2.9 0.029 ± 0.007 1.4 ± 0.5 5-FdUrd (3) 0.0011 ± 0.0002 3.0 ± 0.10.022 ± 0.006 3.0 ± 0.4 0.050 ± 0.011 1.4 ± 0.4 FU 0.33 ± 0.17 0.32 ±0.31 0.18 ± 0.02 18 ± 5  0.54 ± 0.12 CPF 382(1) 0.0255 37.8 0.346 32.7CPF 382(2) 0.0271 39.3 0.21 29.2 CPF 437 36 ±5  >100 >100 >100 >100 >100 CPF 438 0.12 ± 0.02 51 ± 9  2.1 ± 0.6 32 ±2  3.7 ± 0.5 72 ± 0  CPF 373 0.015 ± 0.007 0.027 ± 0.004 0.089 ± 0.0430.32 ± 0.07 CPF 373(2) 0.0061 ± 0.0043 0.064 ± 0.028 0.059 ± 0.046 0.74± 0.18 0.046 ± 0.010 0.74 ± 0.63 0.065 ± 0.013 2.5 ± 1.3 CPF 381 0.028 ±0.007 13 ± 8  0.18 ± 0.03 22 ± 7  CPF 383 0.13 ± 0.04 0.94 ± 0.18 0.64 ±0.57 4.1 ± 2.0 0.92 ± 0.11 14 ± 0  0.48 ± 0.19 9.8 ± 1.4 CPF 384 0.076 ±0.022 1.1 ± 0.1 0.36 ± 0.25 13 ± 1  1.0 ± 0.1 30 ± 10 0.71 ± 0.15 25 ±11 CPF 386 0.031 ± 0.005 0.36 ± 0.01 0.25 ± 0.04 1.6 ± 0.2 0.22 ± 0.042.8 ± 0.0 CPF 393 0.017 ± 0.003 0.18 ± 0.05 0.23 ± 0.04 4.8 ± 0.7 CPF394 0.039 ± 0.001 4.6 ± 0.0 0.65 ± 0.16 22 ± 1  CPF 395 0.011 ± 0.0050.13 ± 0.04 0.16 ± 0.02 2.4 ± 0.8 CPF 396 0.064 ± 0.008 0.82 ± 0.16 0.36± 0.05 6.9 ± 1.8 CPF 508 0.039 ± 0.001 0.14 ± 0.02 0.18 ± 0.00 0.17 ±0.07 0.18 ± 0.05 CPF 509 0.043 ± 0.023 0.15 ± 0.00 0.31 ± 0.06 0.057 ±0.055 0.090 ± 0.014 CPF 576 1.1 ± 0.5 35 ± 8  0.80 ± 0.28 46 ± 14 0.67 ±0.03 27 ± 6  CPF 577 0.21 ± 0.08 25 ± 8  0.89 ± 0.35 32 ± 9  1.2 ± 0.026 ± 1  CPF 578 0.014 ± 0.003 0.088 ± 0.038 0.073 ± 0.018 1.5 ± 0.30.069 ± 0.003 1.5 ± 0.6 CPF 579 0.017 ± 0.007 0.12 ± 0.06 0.059 ± 0.0171.1 ± 0.2 0.068 ± 0.001 1.4 ± 0.4 CPF 580 0.038 ± 0.014 27 ± 6  0.11 ±0.02 43 ± 12 0.13 ± 0.04 15 ± 7  CPF 581 0.0028 ± 0.0010 0.13 ± 0.130.015 ± 0.006 0.28 ± 0.04 0.029 ± 0.023 0.44 ± 0.35 CPF 582 0.031 ±0.010 0.13 ± 0.02 0.035 ± 0.025  0.92 ± 0.007 0.071 ± 0.036 2.2 ± 1.3CPF 583 0.35 ± 0.07 31 ± 5  0.98 ± 0.40 28 ± 8  1.1 ± 0.4 20 ± 11 CPF585 0.016 ± 0.006 0.062 ± 0.009 0.053 ± 0.021 0.19 ± 0.04 0.078 ± 0.0181.3 ± 0.9 CPF 586 0.073 ± 0.035 4.1 ± 1.2 0.28 ± 0.03 25 ± 0  0.15 ±0.02 11 ± 7  CPF 587 0.012 ± 0.007 5.6 ± 1.3 0.10 ± 0.03 7.2 ± 0.1 0.16± 0.08 6.8 ± 1.5 CPF 588 0.27 ± 0.11 1.2 ± 0.7 0.49 ± 0.05 6.7 ± 1.00.70 ± 0.11 32 ± 26 CPF 589 0.022 ± 0.004 0.11 ± 0.06 0.064 ± 0.007 0.84± 0.60 0.12 ± 0.02 2.7 ± 1.5Assay 2.

Samples were also assayed for their % retention of activity inmycoplasma infected cells. The results are set out in Table 2 below. Theresults show that compounds of the present invention can retain highactivity in mycoplasma infected cells, in contrast to the activity shownby 5-FdURD. Administration of a Thymidine Phosphorylase (TP) inhibitorrestores the cytostatic activity of 5-FdUrd in myocoplasma infected cellcultures, providing evidence of the deteriorating role of TP in theeventual cytostatic activity of 5-FdUrd. As mycoplasma infection ofcells is known to greatly reduce the activity of nucleosides, including5-FdUrd, the activity of some nucleosides in mycoplasma infected cellsprovides a potential benefit in patients that are mycoplasma infected.

TABLE 2 CC50 values in μM for 5-FdUrd and compounds embodying thepresent invention in mycoplasma negative and positive cells, and %retention of activity on mycoplasma infection. “% retention” is ameasure of the ratio of the CC50 values measured with respect to L1210with respect to those for L1210/Hyor and is calculated as CC50_(L1210) ×100 ÷ CC50_(L1210/Hyor). Cpd L1210 L1210/Hyor % Retention 5-FdUrd0.00051 0.278 0.2 CPF 373 0.011 0.025 44 CPF 381 0.026 0.15 18 CPF 3930.029 0.02 145 CPF 394 0.030 0.26 12 CPF 395 0.019 0.045 42 CPF 3960.056 0.17 33 CPF 576 1.4 2.73 51 CPF 577 0.23 0.63 36 CPF 578 0.0150.048 31 CPF 579 0.019 0.045 42 CPF 580 0.048 0.41 12 CPF 581 0.00370.017 22 CPF 582 0.035 0.042 83 CPF 583 0.387 11.9 3.3 CPF 585 0.0210.051 41 CPF 586 0.1 0.87 11 CPF 587 0.022 4.2 0.5 CPF 588 0.237 0.39 61CPF 589 0.02 0.063 32

Further experiments (Assays 3 to 8 below) were carried out with respectto the compound CPF 373 embodying the present invention.

Assay 3. Cytostatic Activity of 5-FdUrd and its Prodrug CPF-373 AgainstTK-Competent and TK-Deficient Tumour Cell Lines

The cytostatic activity of 5-FdUrd and CPF-373 was determined indifferent TK-expressing and TK-deficient tumour cell lines. As shown inTable 3, 5-FdUrd is strongly dependent on the expression of TK for itscytostatic activity. Its IC₅₀ increased by 4,000-fold for L1210/TK⁻cells (IC₅₀: 3.1 μM) versus wild-type L1210/0 cells (IC₅₀: 0.0008 μM)and by 50-fold for CEM/TK⁻ cells (IC₅₀: 1.5 μM) versus CEM/0 cells(IC₅₀: 0.028 μM). In contrast, the cytostatic activity of the 5-FdUrdprodrug CPF-373 remained virtually unchanged in TK-deficient cells whencompared with wild-type cells (IC₅₀: 0.027 and 0.011 μM for L1210/TK⁻and L1210/0, and 0.32 and 0.089 μM for CEM/TK⁻ and CEM/0 cells,respectively). Although the cytostatic activity of CPF-373 was 3- to10-fold inferior to 5-FdUrd against wild-type L1210/0 and CEM/0 cells,it proved 5- to 100-fold superior to 5-FdUrd in the TK-deficient tumourcell lines (see Table 3).

TABLE 3 Cytostatic activity of 5-FdUrd and CPF-373 as represented by theIC₅₀ value in different cell lines IC₅₀ ^(a) (μM) Cell lines 5-FdUrdCPF-373 L1210/0  0.0008 ± 0.000095 0.011 ± 0.0065 L1210/TK− 3.1 ± 0.140.027 ± 0.0028 L1210.Hyor 0.24 ± 0.054 0.025 ± 0.0073 CEM/0 0.028 ±0.0014 0.089 ± 0.030  CEM/TK−  1.5 ± 0.071 0.32 ± 0.049 U87 0.007 ±0.001  0.035 ± 0.0005 U87.Hyor 3.0 ± 0.55 0.039 ± 0.0025 ^(a)50%Inhibitory concentration or compound concentration required to inhibittumour cell proliferation by 50%Assay 4. Effect of Mycoplasma Infection of Tumour Cell Cultures on theCytostatic Activity of 5-FdUrd and its Prodrug CPF-373

The L1210/0 cell cultures were infected with the mycoplasma species M.hyorhinis (cells designated: L1210.Hyor). 5-FdUrd markedly lost itscytostatic activity against the mycoplasma-infected L1210.Hyor cells by300-fold (IC₅₀: 0.24 μM). Also, 5-FdUrd lost its cytostatic activity by400-fold in U87.Hyor cell cultures when compared with uninfected U87cells (see Table 3). In sharp contrast, the 5-FdUrd prodrug CPF-373 kepta similar cytostatic potential in both L1210/0 and L1210.Hyor cellcultures (IC₅₀: 0.011 and 0.025 μM, respectively). A similar observationwas made for this prodrug when evaluated for its cytostatic activity inU87 and U87.Hyor cell cultures (IC₅₀: 0.035 and 0.039 μM, respectively).Thus, whereas the free nucleoside 5-FdUrd markedly lost its cytostaticpotential against Mycoplasma hyorhinis-infected tumour cell lines, theantiproliferative potential of its prodrug CPF-373 was independent ofthe mycoplasma infection.

Assay 5.

Experiments were carried out to assess the stability of CPF 373 in thepresence of Thymidine Phosphorylase (TP). The experiments areillustrated with reference to FIGS. 9 to 11, each of which comprises NMRspectra, as discussed below. The present assay shows that theinsensitivity of compounds embodying the present invention to the actionof the catabolic enzyme TP, which is often upregulated in tumours,renders the compounds of the present invention more independent of thecatabolic enzyme TP than 5-FdUrd.

Phosphorylase Assay on 5-FdUrd and its ProTide Compound CPF 373 byThymidine Phosphorylase (TP) Purified from Escherichia Coli.

Nucleoside 5-FdUrd can be degraded to its relative base 5FU by aphosphorolytic reaction, using thymidine phosphorylase purified fromEscherichia coli as well as uridine phosphorylase purified from Ehrlichascite tumor. This breakdown has been suggested to be one of the reasonsfor the limited therapeutic effectiveness of 5-FdUrd according to thefollowing scheme:

The phosphorylase assay was carried out towards phosphorolysis byThymidine Phosphorylase purified from Escherichia coli using in situ ¹⁹FNMR. The application to the ProTide compound CPF 373 was an attempt toprevent the cleavage of the structure and thus circumvent the action ofthe enzyme.

Two potassium phosphate buffers at pH=7.4, 200 nM solution and 300 nMsolution respectively, were used as phosphate donor. Units of enzymewere defined as the amount of enzyme required to hydrolyse about 0.25 mgof inosine per min used as standard. Assays were conducted for 30minutes.

Phosphorylase Assay on 5-FdUrd

Initially, ¹⁹F NMR (470 MHz) spectra of 5-FdUrd and 5FU previouslydissolved in deuterated methanol, were recorded. 5-FdUrd showed asinglet at −δ−167.21 ppm and 5FU at −δ−169.30 ppm. Thus, thephosphorylase assay was carried out by dissolving 5-FdUrd in deuteratedmethanol, in the presence of potassium phosphate buffer (200 nMsolution; pH=7.4), recording the blank before of the addition of theenzyme thymidine phosphorylase (TP) (20.7 UNI). ¹⁹F NMR spectra,recorded at 25° C., showed the singlet of 5-FdUrd at −δ−165.17 ppm and anew peak at −δ−169.50 ppm, attributed to 5FU, as shown in FIG. 9 atspectrum A.

Then, to prove the cleavage of the nucleoside into the relative base, anew experiment was performed by dissolving equal moles of the nucleosideanalogue 5-FdUrd and the relative base 5FU, at the same conditiondescribed above without the TS enzyme, as shown in FIG. 9 at spectrum B.This spectrum showed two singlets with the same chemical shiftspreviously observed in FIG. 9 spectrum A. These data confirmed that the5FU has a chemical shift at −δ−169.50 ppm and thus the phosphorolyticaction of enzyme (TP). Conversion of nucleoside 5-FdURd into the freebase 5FU was 66%.

When the initial concentration of potassium phosphate buffer wasincreased from 200 nM up to 205 nM, substrate 5-FdUrd was fullyconverted into the base 5-FU as shown in the FIG. 10.

Phosphorylase Assay on ProTide Compound CPF 373

Phosphorylase assay was applied to benzyl L-alanine phenyl derivativeCPF 373 in order to investigate the stability, following the procedureand the conditions above described. ProTide compound CPF 373 proved tobe completely stable as showed by comparing chemical shifts of sampleanalysed without TP enzyme, as shown in FIG. 11 spectrum A, and in thepresence of TP, as shown in FIG. 11 spectrum B. ¹⁹F NMR was repeatedafter 4 days and the ProTide compound CPF 373 was shown once again to bestable.

These experiments confirmed that the nucleoside 5-FdUrd is rapidlydegraded into its relative base 5FU by a phosphorolytic reaction, in thepresence of thymidine phosphorylase, with a half-life of less than 30minutes, while prodrug compound CPF 373 showed an evident stabilityagainst TP enzymatic activity, at longer time exposure up to 3 days.This important result showed that 5-FdUrd ProTides derivatives embodyingthe present invention could favor the therapeutic effect of 5-FdUrd.

Assay 6. Exposure of 5-FdUrd and CPF-373 to E. coli-Encoded TP andHuman-Encoded TP and UP

The substrate specificity of thymidine phosphorylase towards naturalthymidine (dThd), uridine (Urd), 5-FdUrd and CPF-373 was investigated byhigh pressure liquid chromatography (HPLC). Reaction mixtures containing100 μM test compound and recombinant TP or UP (human TP: 8.6 ng/μL; E.coli TP: 3.0 ng/μL; human UP: 4 ng/mL) in a total volume of 500 μLreaction buffer (10 mM TrisHCl; 300 μM NaCl; 1 mM EDTA; 2 mMKH₂PO₄/K₂HPO₄) were incubated at room temperature. At different timepoints (i.e. 0, 20, 40 min) 100 μL aliquots of the reaction mixtureswere withdrawn and heated at 95° C. for 3 min to inactivate the enzyme.The resulting reaction products were separated on a reverse-phase RP-8column (Merck, Darmstadt, Germany) and quantified by HPLC analysis(Alliance 2690, Waters, Milford, Mass.). The separation of dThd fromthymine was performed by a linear gradient from 98% separation buffer(50 mM NaH₂PO₄ and 5 mM heptane sulfonic acid, pH 3.2) and 2%acteonitrile, to 20% separation buffer+80% acetonitrile (8 min 98%separation buffer+2% acetonitrile; 5 min linear gradient of 98%separation buffer+2% acetonitrile to 20% separation buffer+80%acetonitrile; 10 min 20% separation buffer+80% acetonitrile, followed byequilibration at 98% separation buffer+2% acetonitrile). UV-baseddetection was performed at 267 nm. The separation of Urd from uracil wasperformed by a linear gradient from 100% separation buffer (see above)to 60% separation buffer+40% acetonitrile (3 min 100% separation buffer;6 min linear gradient of 100% separation buffer to 60% separationbuffer+40% acetonitrile; 6 min 60% separation buffer+40% acetonitrile,followed by equilibration at 100% separation buffer). UV-based detectionwas performed at 258 nm.

Phosphorolysis of 5-FdUrd and CPF-373 by Thymidine and UridinePhosphorylases

5-FdUrd and its prodrug CPF-373 were exposed to purified thymidinephosphorylase derived from E. coli or human erythrocytes, and topurified uridine phosphorylase derived from human tumors. Whereas E.coli and human TP rapidly converted dThd and 5-FdUrd to their freebases, CPF-373 kept fully stable in the presence of these enzymes (FIG.2). Under similar experimental conditions, uridine was converted touracil by human UP, but not by E. coli TP, or human TP. When bothcompounds were exposed to UP, dThd and CPF-373 were not affected by theenzyme, whereas 5-FdUrd was slightly hydrolysed (FIG. 2, panel C).

Assay 7. Thymidylate Synthase (TS) Activity Measurements

The activity of TS in intact L1210/0 and L1210/TK⁻ cells was measured byevaluation of tritium release from [5-³H]dUMP (formed in the cells from[5-³H]dUrd or [5-³H]dCyd) in the reaction catalysed by TS. This methodhas been described in detail by Balzarini & De Clercq (1984). Shortly,cell cultures (500 μL DMEM culture medium) were prepared containing˜3×10⁶ L1210 cells and appropriate amounts of the test compounds(5-FdUrd and CPF-373). After 30 min, 2 h and 4 h preincubation of thecells with the compounds at 37° C., 1 μCi of [5-³H]dUrd or [5-³H]dCydwas added to the cell cultures. After 30 min incubation, 100 μL of thecell suspensions were withdrawn and added to a cold suspension of 500 μLactivated charcoal (VWR, Haasrode, Belgium) (100 mg/ml in TCA 5%). After10 min, the suspension was centrifuged at 13,000 rpm for 10 min, afterwhich the radioactivity in 400 μL supernatant was counted in a liquidsinctillator using OptiPhase HiSafe (Perkin Elmer, Waldham, Mass.).

Inhibition of Thymidylate Synthase (TS) by 5-FdUrd and CPF-373

The major target for the cytostatic activity of 5-FdUrd is thymidylatesynthase (TS). The activity of TS in intact tumour cells can be directlymonitored by measuring the tritium release in intact L1210/0 cellcultures that were exposed to [5-³H]deoxyuridine ([5-³H]dUrd) or[5-³H]deoxycytidine ([5-³H]dCyd). Indeed, after intracellular conversionof [5-³H]dUrd or [5-³H]dCyd to [5-³H]dUMP, the C-5 tritium atom on thepyrimidine base is released during the TS-catalysed reductivemethylation. The ability of 5-FdUrd and its prodrug CPF-373 to inhibittritium release from [5-³H]dUrd and [5-³H]dCyd was therefore evaluatedin L1210/0 cell cultures at a variety of compound concentrations.5-FdUrd proved to be a potent inhibitor of TS in situ. Its IC₅₀ fortritium release from [5-³H]dCyd and [5-³H]dUrd was around 0.0007-0.0009μM (see Table 4).

TABLE 4 IC₅₀ values of 5-FdUrd and CPF-373 against TS in intact L1210/0tumour cells (as determined by tritium release from [5-³H]dUrd and[5-³H]dCyd after 30 min exposure to the drugs). IC₅₀ ^(a) (μM) CompoundTritium release from dUrd* Tritium release from dCyd* 5-FdUrd 0.0009 ±0.0002 0.0007 ± 0.003 CPF-373 0.16 ± 0.05  0.19 ± 0.08 ^(a)50%Inhibitory concentration or compound concentration required to inhibittritium release from [5-³H]dUrd or [5-³H]dCyd in drug-exposed L1210/0cell cultures by 50%.

The inhibitory activity of CPF-373 on tritium release was much less(˜200-fold) pronounced than that of 5-FdUrd, especially after only 30min preincubation of the cells with the drugs (IC₅₀: 0.16-0.19 μM).However, longer preincubation times of the cells (up to 4 hr) with5-FdUrd and CPF-373 before measuring TS activity in the intact tumourcells revealed a much more pronounced inhibitory activity of the prodrugagainst TS in situ (FIG. 3). Indeed, whereas the inhibition of ³Hrelease was only 2-fold increased upon longer preincubation times of5-FdUrd, the inhibitory potential of CPF-373 increased 10-fold (FIG. 3,panels A and B, and C and D).

Preincubation of the tumour cells with 5-FdUrd and CPF-373 for at least4 hrs results in TS inhibition in the intact tumour cells at drugconcentrations that are very comparable with the 50% cytostatic activityconcentrations of these drugs.

The present observations thus indicate that the 5-FdUrd prodrug needsseveral metabolic conversion steps before reaching TS as the targetenzyme for inhibition, and support the view that CPF-373 acts as anefficient prodrug of 5-FdUrd to exert its eventual cytostatic activity.

The activity of TS in the presence of 5-FdUrd and CPF-373 was alsomeasured in intact L1210/TK⁻ cells using [5-³H]dCyd as an externallysupplied substrate (due to TK deficiency, [5-³H]dUrd cannot be used). Asdemonstrated in Table 5 and FIG. 3 (panels E and F), the concentrationof 5-FdUrd required to cause 50% inhibition of TS decreased by a factor5,700 in TK-deficient L1210/TK⁻ cells (IC₅₀: 1.4 μM) when compared towild-type L1210/0 cells (IC₅₀: 0.0003 μM). In contrast, the inhibitoryactivity of CPF-373 against TS remained virtually unchanged in L1210/TK⁻cells (IC₅₀: 0.053 μM in L1210/TK⁻ cells versus 0.013 μM in L1210/0cells).

TABLE 5 IC₅₀ values of 5-FdUrd and CPF-373 against TS in intact L1210/0and L1210/TK⁻ cells (as determined by tritium release from [5-³H]dCydafter 4 hours of preincubation with the products) IC₅₀ ^(a) (μM)Compound L1210/0 L1210/TK⁻ 5-FdUrd 0.0003 ± 0.00003 1.42 ± 0.09 CPF-3730.013 ± 0.008   0.053 ± 0.0009 ^(a)50% Inhibitory concentration orcompound concentration required to inhibit tritium release from[5-³H]dCyd in drug-exposed L1210 cells by 50% upon pre-exposure of thetumour cells for 4 hrs to the drugs.Assay 8. Stability AssaysCarboxypeptidase Y (EC 3.4.16.1) Assay

The enzymatic stability of the prodrug CPF-373 towards carboxypeptidaseY was studied using in situ ³¹P NMR (202 MHz). The experiment wascarried out by dissolving CPF-373 (3.0 mg) in d₆-acetone (150 μL) andadding TRIZMA buffer pH 7.6 (300 μL). The resulting solution was placedin an NMR tube and a ³¹P-NMR experiment at 25° C. was recorded as theblank experiment. The enzyme Carboxypeptidase Y (0.2 mg) was solubilisedin TRIZMA (150 μL) and added to the solution of the phosphoramidatederivative in the NMR tube. Next, the tube was placed in the NMRmachine, which was set to run a ³¹P-NMR experiment (64 scans) every 4minutes for 14 hours at 25° C. Data were processed and analysed with theBruker Topspin 2.1 program. Carboxypeptidase Y and TRIZMA buffer werepurchased from Sigma-Aldrich.

Human Serum

The stability of the prodrug CPF-373 in the presence of human serum wasstudied using in situ ³¹P NMR (202 MHz). The ProTide CPF-373 (1) (5.0mg) was dissolved in DMSO (0.05 mL) and D₂O (0.15 mL). Then the samplewas transferred into an NMR tube, which was inserted in the NMR chamberat 37° C. (with enough solvent to obtain a control NMR reading of theblank). Then 0.3 ml human serum was quickly added to the sample in theNMR tube. NMR experiments were programmed to record data every 15minutes for 12 hours and 30 minutes. Because of excess noise and poorshimming profiles (most likely due to the biological media andconcentration), individual spectra were further processed. After normalFourier transform processing, each spectrum was deconvoluted(Lorentz-Gauss deconvolution) to reveal solely the frequency and area ofspectral peaks without the baseline. Data recorded were processed andanalysed with the Bruker Topspin 2.1 program.

Buffer at pH 1

The stability of the prodrug CPF-373 towards hydrolysis at pH=1 wasstudied using in situ ³¹P NMR (202 MHz). The ProTide CPF-373 (1) (2.6mg) was dissolved in MeOD (0.1 mL) after which 0.5 mL buffer (pH=1)(prepared from equal parts of 0.2 M HCl and 0.2 M KCl) was added. Thenthe sample was transferred into an NMR tube, and a ³¹P NMR experimentwas performed at 37° C. recording the data every 12 minutes for 14hours. Data were processed and analysed with the Bruker Topspin 2.1program.

Buffer at pH 8

The stability of the prodrug CPF-373 towards hydrolysis at pH=8 wasstudied using in situ ³¹P NMR (202 MHz). The ProTide CPF-373 (1) (4.9mg) was dissolved in MeOD (0.1 mL) after which 0.5 mL buffer (pH=8)(prepared from a solution of 0.1 M Na₂HPO₄, which was adjusted by 0.1 MHCl) was added. Then the sample was transferred into an NMR tube, and a³¹P NMR experiment was performed at 37° C. recording the data every 12minutes for 14 hours. Data were processed and analysed with the BrukerTopspin 2.1 program.

Stability Studies

Chemical stability studies on the prodrug CPF-373 (1) have beenperformed by exposing the compound to human serum and to aqueous buffers(pH 1.0 and 8.0) using in situ ³¹P NMR. Each experiment has been carriedout dissolving the ProTide in the suitable deuterated solvent andanalysing the samples at 37° C. for about 14 hours, acquiring scans atthe regular time intervals. For a better resolution original spectra(lower graphs) and deconvoluted ones (upper graphs) are reported. Thestability assay of the phosphoramidate CPF-373 (1), after incubation inhuman serum, showed 73% of unchanged compound after 12 hours and 30minutes as shown in FIG. 6.

The spectra displayed a singlet peak inherent to the human serum at˜δ2.00 and the double peak of the parent at ˜δ4.50 which after 4 hoursand 15 minutes was hydrolyzed to the aminoacyl phosphoramidateintermediate shown as a singlet peak at δ7.20.

When chemical hydrolysis was evaluated at extreme experimentalconditions, i.e. at pH 1.0 and pH 8.0 at 37° C., a full stability ofprodrug CPF-373 (1) in both acidic and basic buffer conditions wasobserved. Spectra were recorded for 14 hours acquiring scans every 12minutes at regular intervals as shown in the FIGS. 7 and 8. The ProTide(1) examined at pH 1.0 showed constant doublet peaks of diastereoisomersat δ4.35; 4.50 throughout the time of the assay (FIG. 7). Also, at pH8.0 the spectra displayed a persistent peak of the prodrug (1) at δ4.48and a singlet peak at δ2.55 corresponding to a buffer peak (FIG. 8).

Metabolism of 5-FdUrd Phosphoramidates

As shown in FIG. 4, the putative mechanism of activation of the ProTidesinside the cell, after uptake, involves a first enzymatic activationstep mediated by a carboxypeptidase-type enzyme which hydrolyzes theester of the aminoacyl moiety (step a) followed by spontaneouscyclization and subsequent spontaneous displacement of the aryl group(step b) and opening of the unstable ring mediated by water (step c).The last step involves a hydrolysis of the P—N bond mediated by aphosphoramidase-type enzyme (step d) with release of the nucleosidemonophosphate in the intact cell (FIG. 4) (McGuigan et al., 2009;Mehellou et al., 2010).

To prove the proposed metabolic scheme for CPF-373 (1) and whether theester motif of the 5-FdUrd phosphoramidate derivative is cleaved-off ornot, an enzyme incubation experiment was carried out that was designedto mimic the first stages of the putative activation in the intacttumour cells. The compound (1) was incubated with carboxypeptidase Y(also known as cathepsin A) in TRIZMA buffer and the conversion of (1)was monitored by ³¹P NMR. Spectra were recorded for 14 h acquiring scansat the periodic intervals every 4 minutes as shown in FIG. 5. For abetter resolution original spectra (lower graphs) and deconvoluted ones(upper graphs) are shown.

At the ³¹P NMR the prodrug CPF-373 (1) appeared as two peaks 64.07; 4.23corresponding with the two diastereoisomers noted as parent with thecharacteristic doubling-up of the chiral phosphate centre of thephosphoramidate. After the addition of cathepsin A the compound wasquickly hydrolyzed after 4 minutes to intermediates δ4.95; 5.16 whichlack the ester motif and this intermediate did not persist as it was inturn quickly metabolized to the aminoacyl phosphoramidate intermediate,the final product in this assay, via the loss of the aryl group (steps ato c in FIG. 4). The intermediate appeared as a singlet peak at δ6.85due to the achiral phosphate centre. Thus, the enzymatic assay spectrashowed a fast metabolism of the parent ˜δ4.00 with complete conversionto the putative intermediate within 26 minutes, which further stayedconsistently present throughout the 14 h of the assay. The cleavage ofthe P—N bond releasing the nucleoside monophosphate was not detected inthe enzyme experiment, as expected. This experiment indicates that thefirst activation step of ProTide CPF-373 (1) may be sufficientlyefficient, and therefore, may allow the eventual delivery of thenucleoside monophosphate metabolite in the intact tumour cells.

CONCLUSION

In conclusion, the present invention provides novel phosphoramidatenucleotide prodrugs of the anticancer nucleoside analogue5-fluoro-2′-deoxyuridine (5-FdUrd), which were synthesized and evaluatedfor their cytostatic activity. Whereas 5-FdUrd substantially lost itscytostatic potential in thymidine kinase (TK)-deficient murine leukaemiaL1210 and human lymphocyte CEM cell cultures, compounds of the presentinvention, for example CPF-373, markedly kept their antiproliferativeactivity in both the wild-type and TK-deficient tumour cells and arethus largely independent of intracellular TK activity to exert theircytostatic action. CPF-373, for example, was found to inhibitthymidylate synthase (TS) in the TK-deficient and wild-type cell linesat drug concentrations that correlated well with its cytostatic activityin these cells. CPF-373 does not seem to be susceptible to inactivationby catabolic enzymes such as thymidine phosphorylase (TP) and uridinephosphorylase (UP). These findings are in line with the observationsthat 5-FdUrd, but not CPF-373, substantially loses its cytostaticpotential in the presence of TP-expressing mycoplasmas in the tumourcell cultures. Therefore, compounds of the present invention such asCPF-373 are novel 5-FdUrd phosphoramidate prodrugs that (i) maycircumvent potential resistance mechanisms of tumour cells (e.g.decreased TK activity) and (ii) is not degraded by catabolic enzymessuch as TP whose activity is often upregulated in tumour cells orexpressed in mycoplasma-infected tumour tissue. Incorporated in byreference in its entirety is Vande Voorde, J. et al BiochemicalPharmacology 82 (2011) 441-452.

Embodiments of the present invention, as set out below, are disclosed inMcGuigan, C. et al J. Med. Chem. 2011, 54 7247-7258 (published Sep. 5,2011), the contents of which in their entirety are hereby incorporatedby reference.

Table 6 below records the cytostatic activity of 5-FU, 5-FdUrd,reference example CPF382 and compounds embodying the present inventionagainst tumour cell lines in terms of IC₅₀ or compound concentrationrequired to inhibit tumour cell proliferation by 50%. Data are the mean(±SD) of at least two to four independent experiments. Table 6identifies the phosphoramidate motif of reference example CPF382 and ofcompounds embodying the present invention with respect to: “aryl”, whichcorresponds to Ar of Formula I and is either phenyl (Ph) or 1-naphthyl(Nap); “ester”, which corresponds to R₃ of Formula I; and “AA”, whichsets out the amino acid whose alpha C atom and substituents on the alphaC atom correspond to CR₁R₂ of Formula I. Table 6 discloses compoundsembodying the present invention not previously mentioned above in Table1, as well as additional data for some of the compounds mentioned inTable 1.

TABLE 6 IC₅₀ (μM) Compd aryl Ester AA L1210/0 L1210/TK⁻ Cem/0 Cem/TK⁻HeLa HeLa/TK⁻ 5-FU 0.33 ± 0.17 0.32 ± 0.31 18 ± 5  12 ± 1  0.54 ± 0.120.23 ± 0.01 5-FdUrd 0.0011 ± 0.0002 3.0 ± 0.1 0.022 ± 0.006 3.0 ± 0.40.050 ± 0.011 1.4 ± 0.4 Ph Me Ala 0.022 ± 0.007 41 ± 3  0.70 ± 0.37 35 ±12 0.28 ± 0.14 4.7 ± 0.4 Ph Et Ala 0.13 ± 0.04 0.94 ± 0.18 0.92 ± 0.1114 ± 0  0.48 ± 0.19 9.8 ± 1.4 Ph i-Pr Ala 0.076 ± 0.022 1.1 ± 0.1 1.0 ±0.1 30 ± 10 0.71 ± 0.15 25 ± 11 Ph c-Hex Ala 0.039 ± 0.001 0.14 ± 0.020.17 ± 0.07  1.2 ± 0.01 0.18 ± 0.05 5.9 ± 0.4 Ph Bn Ala 0.028 ± 0.007 13± 8  0.18 ± 0.03 22 ± 7  0.13 ± 0.01 19 ± 2  Ph Et Val 0.16 ± 0.05 42 ±2  1.0 ± 0.1 >250 1.2 ± 0.3 27 ± 7  Ph Bn Leu 0.044 ± 0.025 2.0 ± 0.30.24 ± 0.04 16 ± 1  0.067 ± 0.042 5.6 ± 0.3 Ph Bn Ile 0.076 ± 0.022 1.1± 0.1 1.0 ± 0.1 30 ± 10 0.71 ± 0.15 25 ± 11 Ph Bn Phe 0.036 ± 0.010 39 ±4  0.25 ± 0.02 11 ± 3  0.014 ± 0.007 12 ± 2  Ph Pnt Met 0.11 ± 0.06 2.2± 0.5 0.35 ± 0.13 13 ± 1  0.15 ± 0.00 7.1 ± 1.2 Ph Bn Met 0.073 ± 0.0354.1 ± 1.2 0.28 ± 0.03 25 ± 0  0.15 ± 0.02 11 ± 7  Ph Bn Pro 0.35 ± 0.0731 ± 5  0.98 ± 0.40 28 ± 8  1.1 ± 0.4 20 ± 11 Ph Et DMG 0.039 ± 0.0014.6 ± 0.0 0.65 ± 0.16 22 ± 1  0.59 ± 0.09 17 ± 2  Ph Bn DMG 0.017 ±0.003 0.18 ± 0.05 0.23 ± 0.04 4.8 ± 0.7 0.24 ± 0.07 3.7 ± 0.1 Nap Et Ala0.031 ± 0.005 0.36 ± 0.01 0.25 ± 0.04 1.6 ± 0.2 0.22 ± 0.04 2.8 ± 0.0Nap Pr Ala 0.021 ± 0.012 0.16 ± 0.07 0.14 ± 0.01 1.1 ± 0.2 0.11 ± 0.032.5 ± 0.1 Nap Butyl Ala 0.022 ± 0.004 0.11 ± 0.06 0.064 ± 0.007 0.84 ±0.60 0.12 ± 0.02 2.7 ± 1.5 Nap Pnt Ala 0.0028 ± 0.0010 0.13 ± 0.13 0.015± 0.006 0.28 ± 0.04 0.029 ± 0.023 0.44 ± 0.35 Nap Hex Ala 0.0072 ±0.0000 0.076 ± 0.015 0.080 ± 0.020 0.65 ± 0.34 0.039 ± 0.018 1.8 ± 0.1Nap c-Bu Ala 0.014 ± 0.003 0.088 ± 0.038 0.073 ± 0.018 1.5 ± 0.3 0.069 ±0.003 1.5 ± 0.6 Nap c-Pnt Ala 0.031 ± 0.010 0.13 ± 0.02  0.35 ± 0.025 0.92 ± 0.007 0.071 ± 0.036 2.2 ± 1.3 Nap c-Hex Ala 0.043 ± 0.023 0.15 ±0.00 0.057 ± 0.055 1.0 ± 0.1 0.090 ± 0.014 ND Nap CH₂-t-Bu Ala 0.27 ±0.11 1.2 ± 0.7 0.49 ± 0.05 6.7 ± 1.0 0.70 ± 0.11 32 ± 26 Nap CH₂CH₂-t-BuAla 0.016 ± 0.006 0.062 ± 0.009 0.053 ± 0.021 0.19 ± 0.04 0.078 ± 0.0181.3 ± 0.9 Nap CH₂-c-Pr Ala 0.017 ± 0.007 0.12 ± 0.06 0.059 ± 0.017 1.1 ±0.2 0.068 ± 0.001 1.4 ± 0.4 Nap 2-Ind Ala 0.021 ± 0.002 40 ± 0  0.079 ±0.018 1.0 ± 0.2 0.10 ± 0.06 7.1 ± 2.1 Nap Bn Ala 0.011 ± 0.007 0.045 ±0.027 0.068 ± 0.035 0.31 ± 0.06 0.065 ± 0.013 2.5 ± 1.3 Nap THP Ala0.038 ± 0.014 27 ± 6  0.11 ± 0.02 43 ± 12 0.13 ± 0.04 15 ± 7  Nap c-HexVal 1.1 ± 0.5 35 ± 8  0.80 ± 0.28 46 ± 14 0.67 ± 0.03 27 ± 6  Nap PntLeu 0.017 ± 0.001 1.2 ± 0.4 0.071 ± 0.008 15 ± 4  0.039 ± 0.014 7.5 ±0.4 Nap Bn Leu 0.028 ± 0.004 1.5 ± 0.6 0.13 ± 0.00 30 ± 6  0.080 ± 0.0229.4 ± 1.4 Nap Pnt Ile 0.22 ± 0.12 12 ± 2  0.46 ± 0.11 17 ± 1  0.30 ±0.02 11 ± 1  Nap Pnt Phe 0.026 ± 0.001 2.9 ± 1.2 0.10 ± 0.00 8.3 ± 1.00.040 ± 0.000 6.6 ± 0.5 Nap Bn Phe 0.012 ± 0.007 5.6 ± 1.3 0.10 ± 0.037.2 ± 0.1 0.16 ± 0.08 6.8 ± 1.5 Nap Bn Met 0.072 ± 0.001 1.9 ± 0.2 0.19± 0.10 11 ± 1  0.087 ± 0.017 8.3 ± 0.0 Nap Bn Pro 0.21 ± 0.08 25 ± 8 0.89 ± 0.35 35 ± 9  1.2 ± 0.0 26 ± 1  Nap Et DMG 0.064 ± 0.008 0.82 ±0.16 0.36 ± 0.05 6.9 ± 1.8 0.20 ± 0.12 3.2 ± 0.0 Nap Pnt DMG 0.037 ±0.010 0.30 ± 0.13 0.14 ± 0.00 5.4 ± 1.1 0.12 ± 0.03 2.3 ± 0.1 Nap Bn DMG0.011 ± 0.005 0.13 ± 0.04 0.16 ± 0.02 2.4 ± 0.8 0.078 ± 0.020 3.1 ± 0.6

Table 7 below records the cytostatic activity of 5-FdUrd, referenceexample CPF382 and compounds embodying the present invention in wildtype murine leukemia L1210 cell cultures (L1210/0) and L1210 cellcultures, infected with Mycoplasma hyorhinis (L1210.Hyor) in terms ofIC₅₀ or compound concentration to inhibit cell proliferation by 50%.Data are mean (±SD) of at least two to four independent experiments.Table 7 identifies the phosphoramidate motif of reference example CPF382and of compounds embodying the present invention, as discussed abovewith respect to Table 6, but with “Naph” standing for 1-naphthyl. Table7 discloses compounds embodying the present invention not previouslymentioned above in Table 2, as well as additional data for some of thecompounds mentioned in Table 2.

TABLE 7 IC₅₀ (μM) IC₅₀(L1210.Hyor)/ Compd aryl ester AA L1210/0L1210.Hyor IC₅₀(L1210/0) 5-FdUrd 0.0009 ± 0.0003 0.34 ± 0.13 378 Ph MeAla 0.040 ± 0.016 0.87 ± 0.28 22 Ph Et Ala  0.11 ± 0.0021 0.54 ± 0.12 5Ph i-Pr Ala 0.050 ± 0.013 0.70 ± 0.10 14 Ph c-Hex Ala  0.032 ± 0.00500.040 ± 0.016 1.25 Ph Bn Ala 0.026 ± 0.008  0.15 ± 0.006 5.8 Ph Et Val 0.20 ± 0.033 4.4 ± 1.1 22 Ph Bn Leu  0.054 ± 0.0021  0.17 ± 0.047 3.2Ph Bn Ile 0.98 ± 0.39  2.2 ± 0.031 2.2 Ph Bn Phe  0.016 ± 0.0014  0.56 ±0.023 35 Ph Pnt Met  0.13 ± 0.0078 0.41 ± 0.21 3.2 Ph Bn Met 0.058 ±0.035 0.76 ± 0.18 13 Ph Bn Pro  0.35 ± 0.022   18 ± 0.71 51 Ph Et DMG 0.030 ± 0.0005 0.26 ± 0.01 8.7 Ph Bn DMG 0.029 ± 0.001  0.02 ± 0.0020.69 Naph Et Ala  0.028 ± 0.0021  0.095 ± 0.0028 3.4 Naph Pr Ala  0.030± 0.00035  0.036 ± 0.0064 1.2 Naph butyl Ala 0.0095 ± 0.0021  0.021 ±0.0071 2.2 Naph Pnt Ala  0.0021 ± 0.00007  0.006 ± 0.0014 2.9 Naph HexAla  0.0032 ± 0.00035  0.0022 ± 0.00028 0.69 Naph c-Bu Ala  0.011 ±0.0014  0.024 ± 0.00014 2.2 Naph c-Pnt Ala  0.016 ± 0.0007 0.024 ± 0.0051.5 Naph c-Hex Ala 0.036 ± 0.017 0.049 ± 0.004 1.4 Naph CH₂—t-Bu Ala0.093 ± 0.033  0.18 ± 0.069 1.9 Naph CH₂ CH₂—t-Bu Ala  0.012 ± 0.0018 0.032 ± 0.0088 2.7 Naph CH₂—c-Pr Ala  0.014 ± 0.0042  0.031 ± 0.00642.2 Naph 2-Ind Ala 0.039 ± 0.019 0.042 ± 0.040 1.08 Naph Bu Ala 0.011 ±0.009 0.025 ± 0.01  2.27 Naph THP Ala  0.041 ± 0.0028 0.48 ± 0.11 11.7Naph c-Hex Val  1.2 ± 0.17 1.29 ± 0.29 1.08 Naph Pnt Leu  0.031 ± 0.00200.035 ± 0.010 1.13 Naph Bn Leu  0.029 ± 0.0021 0.048 ± 0.020 1.7 NaphPnt Ile  0.42 ± 0.021  0.70 ± 0.074 1.67 Naph Pnt Phe  0.030 ± 0.0039 0.14 ± 0.007 4.67 Naph Bn Phe  0.021 ± 0.0061  0.23 ± 0.078 11 Naph BnMet 0.054 ± 0.013  0.20 ± 0.098 3.7 Naph Bn Pro  0.26 ± 0.055  0.65 ±0.070 2.5 Naph Et DMG 0.056 ± 0.04  0.17 ± 0.03 3 Naph Pnt DMG  0.045 ±0.0021  0.019 ± 0.0028 0.42 Naph Bn DMG 0.019 ± 0.004 0.045 ± 0.004 2.4

Table 8 below records the cytostatic activity of 5-FdUrd and compoundsembodying the present invention in CEM cell cultures containing(Cem/hEnt-1) or lacking (Cem/hEnt-0) the hEnt1 transporter in terms ofIC₅₀ or compound concentration required to inhibit tumour cellproliferation by 50%. Data are mean (±SD) of at least two to fourindependent experiments. Table 8 identifies the phosphoramidate motif ofcompounds embodying the present invention, as discussed above withrespect to Table 6, but with “Naph” standing for 1-naphthyl. The data ofTable 8 show that compounds embodying the present invention are lessdependent on the presence of the hENT1 transporter, than 5-FdUrd, sincethey lose only 7- to 15-fold antiproliferative activity against thehENT1-deficient CEM cells. These observations are in agreement with anonly 2- to 7-fold decreased cytostatic activity of compounds embodyingthe present invention in the presence of transport inhibitors (i.e.dipyridamole and NBMPR), compared to a 20- to 60-fold loss ofantiproliferative activity of 5-FuDrd and FdUMP under similarexperimental conditions.

TABLE 8 IC₅₀ (μM) Cem/ Cem/ Cem/hEnt-1 + Cem/hEnt-1 + compd aryl EsterAA hEnt-1 hEnt-0 dipyridamole NBMPR 5-FdUMP 0.05 ± 0.02 3.6 ± 0.69 1.741.06 5-FdUrd 0.04 ± 0.02 2.5 ± 0.65 1.36 0.80 Ph Bn Ala 0.13 ± 0.05 1.4± 0.65 0.66 0.72 Ph Et DMG 0.37 ± 0.14 5.8 ± 0.50 2.35 2.56 Ph Bn DMG0.17 ± 0.06 1.2 ± 0.11 0.26 0.61 Naph Bn Ala 0.05 ± 0.02 0.6 ± 0.11 0.130.26 Naph Et DMG 0.21 ± 0.07 1.4 ± 0.20 0.52 0.62 Naph Bn DMG 0.05 ±0.03 0.4 ± 0.13 0.16 0.28

Studies were performed on compound CPF 381 as follows:

An enzymatic phosphorylase assay was carried out using thymidinephosphorylase (TP, purified from Esherichia coli) in the presence ofpotassium phosphate buffer (300 nM solution, pH 7.4). The ¹⁹F NMRspectrum after 5 min, 14 h and 72 h did not show any evidence ofphosphorolysis. In contrast to 5-FdUrd, CPF 381 is at best a very poor,if any, substrate for thymidine phosphorylase.

A chemical hydrolysis was evaluated under experimental conditions at pH1 and pH 8 and monitored by ³¹P NMR. During the assay (14 h) underacidic conditions (pH 1) only two peaks representing the twodiastereoisomers were recorded. Lack of formation of new signals in the³¹P NMR spectrum indicates that compound CPF 381 is highly stable inacidic medium. The same result was observed when compound CPF 381 wassubjected to the assay under mild basic conditions (pH 8).

Studies were performed on compound CPF 581 as follows:

A enzymatic study using a carboxypeptidase Y assay was performed inwhich compound CPF 581, carboxypeptidase Y, and Trizma buffer (pH 7.6)were dissolved in acetone-d₆ and ³¹P NMR spectrum (202 MHz) spectra wererecorded at regular intervals (every 7 min) over 14 h. Compound CPF 581was rapidly hydrolyzed to a first metabolite lacking the ester (R₃)moiety, both diastereoisomers being processed at roughly similar rate.Further processing of the first metabolite led to the formation of ananionic second metabolite, lacking Ar, within about 45 min with anestimated half life of less than 5 min. The rate of the initialactivation step might thus be considered in general as one ofrequirements for good biological activity of phosphoramidates. Chemicalhydrolysis of compound CPF 373 in the presence of triethylamine andwater produced the diammonium salt of the anionic second metabolite,which was added to the final assay sample derived from compound CPF 373,i.e. containing only the enzymatic second metabolite derived fromcompound CPF 581 in Trizma. The sample had a ³¹P NMR spectrum showingonly a single peak at δ_(P) 6.85 ppm, strongly supporting this part ofthe metabolic pathway and activation of the phosphoramidate compounds ofthe present invention.

Studies were performed on compound CPF 386 as follows:

The stability of compound CPF 386 in the presence of human serum wasinvestigated using in situ ³¹P NMR. A control ³¹P NMR data of compoundCPF 386 in DMSO and D₂O were recorded. The NMR sample was then treatedwith human serum and immediately subjected to further ³¹P NMRexperiments at 37° C. The ³¹P NMR data were recorded every 15 minutesover 14 h. The spectra displayed a single peak inherent to human serumat ˜δ_(P) 2.00 ppm and two peaks corresponding to compound CPF 386 at˜δ_(P) 4.59 and 4.84 ppm. After about 6 h and 45 min the compound washydrolyzed partly to an intermediate, lacking R₃ (Et), as a single peakat δ_(P) 5.79 ppm. After 11 h and 30 min, the formation of the secondmetabolite, lacking Ar (1-naphthyl), shown as single peak at δ_(P) 7.09ppm was observed. After 13 h and 30 min the reaction mixture contained96% of the parent compound CPF 386 together with the proposed firstmetabolite (3%) and second metabolite (1%).

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The invention claimed is:
 1. A method of treatment of cancer comprisingadministering to a Homo sapiens patient in need of such treatment aneffective dose of a compound of formula (I):

wherein Ar is a fused bicyclic aryl moiety or a monocyclic aryl moiety,either of which aryl moieties is carbocyclic or heterocyclic and isoptionally substituted; R₃ is alkyl, which is optionally substituted; R₄is H or alkoyl; R₁ and R₂ are independently selected from the groupconsisting of H and alkyl, or R₁ and R₂ together form an alkylene chainso as to provide, together with the C atom to which they are attached, acyclic system, or one of R₁ and R₂ comprises an alkylene chain attachedto N, the H atom attached to N is absent and one of R₁ and R₂ comprise Hor alkyl, any of which said alkyl moieties or alkylene chains may besubstituted; or a pharmaceutically acceptable derivative or metaboliteof formula I, wherein the compound is not a compound having, incombination, Ar as unsubstituted phenyl, R₃ as CH, R₄ as H, one of R₁and R₂ as H and one of R₁ and R₂ as CH, and wherein the patient hasdeveloped or has the potential to develop resistance in tumour cellswith respect to the activity of 5-fluoracil (5-FU) or5-fluoro-2′-deoxyuridine (5-FdUrd) and wherein the cancer is selectedfrom gastrointestinal cancer, leukemia, lymphoma, pancreatic cancer,prostate cancer, lung cancer, breast cancer, cervical cancer, head andneck cancer, and ovarian cancer.
 2. The method according to claim 1wherein the cancer is selected from the group comprising leukemia,pancreatic, prostate, lung, breast and cervical cancer.
 3. The methodaccording to claim 1 wherein the cancer is selected from the groupcomprising: gastrointestinal cancer, including gastric, small intestine,oesophageal, stomach, colon and rectum cancer; head and neck cancer; andovarian cancer.
 4. The method according to claim 1 wherein the patienthas cells with a lowered level of nucleoside transporter proteins. 5.The method according to claim 1 wherein the patient has nucleosidekinase-deficient cells.
 6. The method according to claim 1 wherein thepatient has mycoplasma-infected cells.
 7. The method according to claim1 wherein the patient has cells with a raised level of thymidylatesynthase (TS).
 8. The method according to claim 1 wherein the patient issusceptible to nucleoside degradation by catabolic enzymes.
 9. Themethod according to claim 8 wherein the catabolic enzymes are selectedfrom the group comprising thymidine phosphorylase, uridine phosphorylaseand deoxycytidine deaminase.
 10. The method according to claim 1 inconjunction with other cancer therapy.
 11. The method according to claim1 wherein Ar is naphthyl.
 12. The method according to claim 11 whereinAr is 1-naphthyl.
 13. The method according to claim 1 wherein Ar isphenyl.
 14. The method according to claim 1 wherein Ar is substituted.15. The method according to claim 1 wherein R₄ is selected from thegroup consisting of H and acetyl.
 16. The method according to claim 15wherein R₄ is H.
 17. The method according to claim 1 wherein R₃ isselected from the group consisting of benzyl and members of the groupcomprising C₁ to C₁₀ alkyls.
 18. The method according to claim 17wherein R₃ is ethyl, n-propyl, n-butyl, n-pentyl, or n-hexyl.
 19. Themethod according to claim 18 wherein R₃ is n-pentyl.
 20. The methodaccording to claim 1 wherein R₁ and R₂ are different and thestereochemistry at the asymmetric centre CR₁R₂ corresponds to a memberselected from the group consisting of an L-amino acid, a D-amino acidand mixtures of L and D amino acids.
 21. The method according to claim 1wherein R₁ and R₂ correspond to the moieties attached to the alpha Catom in a natural occurring alpha amino acid.
 22. The method of claim 21wherein the natural occurring alpha amino acid is L-alanine.
 23. Themethod according to claim 1 wherein Ar is 1-naphthyl, R₃ is benzyl, andone of R₁ and R₂ is H, one of R₁ and R₂ is methyl and the C atom towhich R₁ and R₂ are attached has L-chirality.
 24. The method accordingto claim 1 wherein Ar is 1-naphthyl, R₃ is n-pentyl, one of R₁ and R₂ isH, one of R₁ and R₂ is methyl and the C atom to which R₁ and R₂ areattached has L-chirality.
 25. The method according to claim 1, whereinthe compound is selected from the group comprising:5-Fluoro-2′-deoxyuridine-5′-O-[phenyl(benzoxy-L-alaninyl)]phosphate (CPF381); 5-Fluoro-2′-deoxyuridine-5′-O-[phenyl(ethoxy-L-alaninyl)]phosphate (CPF383);5-Fluoro-2′deoxyuridine-5′-O-[phenyl(isopropoxy-L-alaninyl)] phosphate(CPF384); 5-Fluoro-2′deoxyuridine-5′-O-[phenyl (cyclohexoxy-L-alaninyl)]phosphate (CPF508);5-Fluoro-2′deoxyuridine-5′-O-[p-nitro-phenyl(ethoxy-L-alaninyl)]phosphate (CPF430); 5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl(benzoxy-L-alaninyl)] phosphate (CPF373); 5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (methoxy-L-alaninyl)] phosphate (CPF385);5-Fluoro-2′ deoxyuridine-5′-O-[1-naphthyl (ethoxy-L-alaninyl)] phosphate(CPF386); 5-Fluoro-2′ deoxyuridine-5′-O-[1-naphthyl(isopropoxy-L-alaninyl)] phosphate (CPF387);5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (cyclohexoxy-L-alaninyl)]phosphate (CPF509); 5-Fluoro-2′deoxyuridine-5′-O-[phenyl(benzoxy-α,α-dimethylglycine)] phosphate (CPF393);5-Fluoro-2′deoxyuridine-5′-O-[phenyl (ethoxy-α,α-dimethylglycine)]phosphate (CPF394); 5-Fluoro-2′ deoxyuridine-5′-O-[1-naphthyl(benzoxy-α,α-dimethylglycine)] phosphate (CPF395);5-Fluoro-2′deoxyuridine-5′-O-[1-naphthyl (ethoxy-α,α-dimethylglycine)]phosphate (CPF396);5-Fluoro-2′-deoxyuridine-5′-O-[phenyl(benzoxy-L-prolinyl)] phosphate(CPF583); 5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl(benzoxy-L-prolinyl)]phosphate (CPF577);5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl(3,3-dimethyl-1-butoxy-L-alaninyl)]phosphate (CPF585);5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(cyclobutoxy-L-alaninyl)]phosphate (CPF578);5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(cyclopropylmethanoxy-L-alaninyl)]phosphate (CPF579);5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(tetrahydropyroxy-L-alaninyl)]phosphate (CPF580);5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(pentoxy-L-alaninyl)]phosphate (CPF581);5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(cyclopentoxy-L-alaninyl)]phosphate (CPF582);5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(2-indanoxy-L-alaninyl)]phosphate (CPF597);5-Fluoro-2′-deoxyuridine-5′-O-[phenyl-(benzoxy-L-methioninyl)] phosphate(CPF586);5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(benzoxy-L-phenylalaninyl)]phosphate (CPF587);5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(2,2-dimethylpropoxy-L-alaninyl)]phosphate (CPF588);5-Fluoro-2′-deoxyuridine-5′-O-[1-naphthyl-(butoxy-L-alaninyl)] phosphate(CPF589); or a pharmaceutically acceptable derivative or metabolitethereof.